Adult ; Bone Screws ; Collateral Ligaments ; injuries ; surgery ; Female ; Humans ; Knee Joint ; surgery ; Male ; Middle Aged ; Tibia ; surgery

OBJECTIVETo investigate the effects of phosphorylated mitogen-activated protein kinases (MAPK), including the phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), the phosphorylated protein p38 (p-p38), the phosphorylated c-Jun N-terminal kinase (p-JNK), on phosgene inhalation-induced lung injury and its relationship with matrix metalloproteinase 9 (MMP-9).

METHODSAccording to the random number table, 30 male Wistar rats were divided into air control group (C), phosgene inhalation group (P), PD98059 (specific inhibitor of ERK1/2) group, SB203580 (specific inhibitor of p38) group, and SP600125 (specific inhibitor of JNK) group, with 6 rats in each group. The number of neutrophils in the bronchoalveolar lavage fluid (BALF) was counted and the lung wet-dry ratio (W/D) was examined. The serum levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were determined with ELISA. The protein expressions of p-ERK1/2, p-p38, p-JNK, and MMP-9 in lung tissue were detected with Western blotting. The mRNA level of MMP-9 in lung tissue was detected with real-time fluorescence quantitative PCR. Data were processed with one-way analysis of variance (among groups) and SNK method (paired comparison).

RESULTSCompared with those of group C [respectively (2.0 ± 0.7)×10(4) /mL and 3.7 ± 0.6], the number of neutrophils and W/D of group P [respectively (10.7 ± 1.4)×10(4) /mL and 7.6 ± 0.4] were increased. The number of neutrophils in group SB203580 and group SP600125 was respectively (8.3 ± 1.1)×10(4), (7.9 ± 1.3)×10(4)/mL, with W/D respectively 6.1 ± 1.4, 6.1 ± 0.9, all of which decreased as compared with those of group P (with P values all below 0.01). Compared with those of group C, the levels of TNF-a, IL-1β, IL-6, and IL-8 of group P were increased, but decreased in group SB203580 and group SP600125 compared with that of group P, though still higher than those of group C, and the differences were statistically significant (P < 0.05 or P<0.01). Protein quantities of p-p38 and p-JNK were higher in group P (respectively 1.19 ± 0.22 and 1.43 ± 0.14) than in group C (respectively 0.76 ± 0.06 and 0.74 ± 0.05). Compared with those of group P, the protein levels of p-ERK1/2 (0.47 ± 0.05) in group PD98059, p-p38 (0.88 ± 0.07) in group SB203580, and p-JNK (0.91 ± 0.07) in group SP600125 were significantly reduced (P < 0.05 or P < 0.01). The protein and mRNA levels of MMP-9 were higher in group P (respectively 2.23 ± 0.18 and 4.93 ± 0.12) than in group C (respectively 1.26 ± 0.14 and 1.80 ± 0.03). The protein and mRNA levels of MMP-9 in group SB203580 (respectively 1.58 ± 0.14 and 2.96 ± 0.09) and group SP600125 (respectively 1.55 ± 0.30 and 3.00 ± 0.13) were lower than those in group P (P < 0.05 or P < 0.01).

CONCLUSIONSThe phosgene inhalation can activate the MAPK signaling protein pathway by increasing expressions of p-p38 and p-JNK, which lead to an up-regulation of MMP-9, and this may contribute to the phosgene inhalation-induced lung injury.

Animals ; Burns, Inhalation ; enzymology ; Cytokines ; metabolism ; Disease Models, Animal ; Flavonoids ; pharmacology ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Phosgene ; Phosphorylation ; Pyridines ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVEThis study aimed to investigate the expression and role of the mitogen activated protein kinases (ERK1/2, P38, JNK) in phosgene induced lung injury in rats in vivo.

METHOD30 male wistar rats were randomized into the group as follows, Gas inhalation control group, Phosgene inhalation group, and the following groups of the inhibitors of MAPK, involving SP600125, PD98059 and SB203580, 6 animals in each group, we copy the model of phosgene-induced lung injury, used the directional flow-inhalation device, the air control group inhaled the air, and the intervention groups were given PD98059 (intraperitoneal injection), SB203580 (hypodermic injection), SP600125 (intravenous) respectively before the inhalation of phosgene. The locations and quantities of three subfamilies of MAPKs (ERK1/2, P38, JNK) and p-MAPKs (p-ERK1/2, p-P38, p-JNK) were analyzed by immunohistochemistry and Western Blot analysis respectively; The histopathological changes of lung tissues, the number of neutrophil cells and the W/D were examined.

RESULTThere were rare p-ERK1/2, p-P38 and p-JNK positive expression in alveolar and airway epithelial cells in control group. while the positive cells increased strikingly in phosgene inhalation groups, these cells involved in this process mainly included alveolar epithelial cells, air way epithelial cells, pleural mesothelial cells, infiltrative inflammatory cells, interstitium fibrocytes. After the intervention of the specific inhibitor, the positive cells decreased. As Western Blot analysis show, Protein quantities of p-P38 and p-JNK were higher in phosgene inhalation groups than those in control group, and the differences were significant (P < 0.05). Protein quantities of p-ERK1/2, p-P38 and p-JNK were lower in intervention groups than phosgene inhalation group, and the differences were significant (P < 0.05, P < 0.01). The lung injury in phosgene inhalation groups was more severer compared with the control group, the typical pathological characters of acute lung injury were discovered, the increase of the number of neutrophil cells and W/D. After the intervention of the specific inhibitor SP600125 and SB203580, the number of neutrophil cells and W/D reduced, and the differences were significant (P < 0.05, P < 0.01).

CONCLUSIONPhosgene inhalation may activate the MAPK signaling pathway, and the expression of the phosphorylation of MAPKs increased, especially the P38 ang JNK. The results may contribute to the lung injury induced by phosgene.

Animals ; Inhalation Exposure ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; metabolism ; pathology ; Lung Injury ; etiology ; metabolism ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Phosgene ; adverse effects ; Rats ; Rats, Wistar ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo compare two models of nonalcoholic hepatocellular steatosis.

METHODSHL-7702 cells were incubated with a mixture of of unsaturated oleate acid or 50% fetal bovine serum to induce fat-overloading. Significant fat accumulation was documented by Oil Red O staining , and intracellular triglyceride levels was detected by triglyceride enzymatic assay.

RESULTSThe results showed that both 0.5 mmol/ml oleate acid and 50% FBS were able to induce nonalcoholic hepatocellular steatosis.

CONCLUSIONA nonalcoholic hepatocellular steatosis was induced by 0.5 mmol/ml oleate acid.

Cell Line ; Fatty Liver ; Hepatocytes ; pathology ; Humans ; Models, Biological

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.
Bioreactors ; microbiology ; Colony Count, Microbial ; Escherichia coli ; genetics ; metabolism ; Hepatitis E virus ; genetics ; Nucleocapsid Proteins ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.
Capsid Proteins ; chemistry ; Hepatitis E virus ; chemistry ; Hydrophobic and Hydrophilic Interactions ; Protein Multimerization ; Protein Structure, Tertiary ; Virus Assembly

OBJECTIVEA prospective study was conducted to probe into the relationship between arterial oxygen partial pressure (PaO2) and brain injury during cardiopulmonary bypass (CPB) in infants with cyanotic congenital heart disease (CHD).

METHODEnrolled in the study were 45 cyanotic infants, who were less than three years old and underwent corrective cardiac surgery from August 1(st), 2010 to January 31(st), 2011 at Guangdong General Hospital. All the infants had a pulse oxygen saturation (SpO2) lower than 85% and were randomly allocated into three groups by a specific computer program. In controlled group 1 (G1 group), PaO2 levels were controlled at 80 - 120 mm Hg (1 mm Hg = 0.133 kPa) during CPB; in controlled group 2 (G2 group), PaO2 levels at 120 - 200 mm Hg during CPB; while in uncontrolled group (G3 group), PaO2 levels were at 200 - 400 mm Hg during CPB. Blood samples were collected just before starting CPB, at the end of CPB, and at 3 h, 5 h, and 24 h after CPB (T1, T2, T3, T4, T5) for the determination of serum concentrations of protein S100β, neuron specific enolase (NSE), and adrenomedullin (ADM) by ELISA.

RESULTProtein S100β rose significantly after starting CPB. In group G3, it reached a peak of (699 ± 139) ng/L by the end of CPB, significantly higher than those in groups G1 and G2 [(528 ± 163) ng/L and (585 ± 155) ng/L], and was positively correlated with PaO2 levels (r = 0.526, P < 0.01). NSE levels of group G1 were continuously rising after starting CPB and reached significantly high levels at 3 h or 5 h after CPB [(12.2 ± 3.4) µg/L and (12.3 ± 3.7) µg/L], while those of group G2 rose significantly during CPB [(10.9 ± 4.8) µg/L] and even higher at 3 h or 5 h after CPB [(12.6 ± 5.1) µg/L and (13.2 ± 5.4) µg/L]. NSE levels of group G3 rose significantly during CPB and maintained at a high level [(12.2 ± 5.7) µg/L] afterwards. There was no significant difference in serum ADM concentrations among different time points in each group and among these three groups. All the infants were discharged from the hospital without any obvious nervous symptom and sign.

CONCLUSIONHigh PaO2 during CPB in infants with CHD might cause an increase of serum protein S100β and NSE, indicating that brain injury might become worse with a higher PaO2 and might be positively correlated with PaO2 during CPB.

Cardiopulmonary Bypass ; Child, Preschool ; Cyanosis ; Female ; Heart Defects, Congenital ; blood ; physiopathology ; surgery ; Humans ; Infant ; Male ; Nerve Growth Factors ; blood ; Oximetry ; Oxygen ; blood ; Partial Pressure ; Phosphopyruvate Hydratase ; blood ; Prospective Studies ; S100 Calcium Binding Protein beta Subunit ; S100 Proteins ; blood ; Serum

This study was purposed to investigate the relationship between the levels of soluble receptor activator of nuclear factor kappa B ligand (sRANKL) and osteoprotegerin (OPG) in serum of the patients with multiple myeloma (MM) and multiple myeloma bone disease (MBD). The serum levels of sRANKL, OPG, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-terminal telopeptide of collagen I (CTP-I) which both are indexes for metabolism of osteoclast (OC) in newly diagnosed MM patients (n=42, experimental group) and healthy persons (n=25, control group) were detected by enzyme-linked immunosorbent assay. The roentgenography was used to determine bone damage in MM patients at the same time. According to these results acquired, the correlation of sRANKL/OPG ratio with levels of TRAP-5b/CTP-I, the incidence and degree of bone destruction were analyzed. The results indicated that the level of sRANKL (median value 9.33 microg/L) increased and level of OPG (median value 4.93 microg/L) decreased and the sRANKL/OPG ratio (2.65) increased significantly in experimental group. Compared with control group, the differences in all the corresponding indicators were statistically significant (p<0.05). The sRANKL/OPG ratio was closely related to levels of TRAP-5b (r=0.512, p<0.05) and CTP-I (r=0.481, p<0.05) in MM patients. After all patients in experimental groups were divided into group with bone destruction (n=29) and without bone destruction (n=13), the sRANKL/OPG ratio in the group with bone destruction was 5.13 and much higher than that in group without bone destruction (1.12) (p<0.05). A close correlation between the sRANKL/OPG ratio and degree of bone destruction (r=0.445, p<0.05) was acquired when all MM patients were divided into three groups according to degree of bone destruction, but no difference between the ratio and clinical classification and International Staging System (ISS) in MM patients was found. It is concluded that the sRANKL/OPG ratio in serum of MM patients is significantly elevated, which may be closely related to increase metabolism of OC along with the incidence and degree of bone destruction. In short, the sRANKL/OPG ratio can be used as a reference index for the diagnosis of MBD.
Adult ; Aged ; Bone Diseases ; blood ; diagnosis ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; blood ; diagnosis ; Osteoprotegerin ; blood ; RANK Ligand ; blood

OBJECTIVETo investigate the image findings of bile duct injury after transcatheter arterial chemoembolization (TACE) for hepatic malignancy.

METHODSDuring the past 3 years, 1240 patients with different hepatic malignancies had undergone a total of 2680 TACE procedures. Eighteen patients (1.4%) developed bile duct injuries from 3 weeks to 3 months after TACE. All of the 18 patients received follow-up CT and ultra-sonography, 14 MRI and 15 digital subtract angiography (DSA). The image data was retrospectively reviewed, with the potential predisposing factors correlated to TACE-induced bile duct injury.

RESULTSTACE-induced bile duct injuries developed in 13 of 148 patients with liver metastasis (8.8%), 5 of 1092 patients with hepatocellular carcinoma (HCC) (0.5%). On image examination, focal peripheral intrahepatic bile duct dilatation was detected in 4 cases, multiple bile duct dilatations with segmental or sub-segmental distribution in 8, and a large lobular cystic lesion or biloma in 6 cases, and progressive atrophy of the corresponding hepatic parenchyma in 6 patients in whom the TACE induced-bile injury developed at different intervals after TACE. The incidence of bile duct injury was higher in non-cirrhotic patients with metastatic liver lesions than in patients with hepatocellular carcinoma associated with cirrhosis (P < 0.01), and it was also higher in patients using an emulsion of lipiodol-cisplatin or carboplatin than in patients using other emboliging agents (P < 0.01). The incidence was higher either in patients with hypovascular lesions than in patients with hypervascular lesions (P < 0.05).

CONCLUSIONBiliary abnormalities, including focal and multiple intrahepatic bile duct dilatation, and cystic lesion or biloma, may develop and can be detected during the follow-up examination imaging in patients with hepatic malignancy after TACE. Noncirrhotic liver and intact function, due to the lack of peri-biliany collateral circulation, are the significant predisposing factors to the development of TACE-induced bile duct injury.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; adverse effects ; Bile Ducts ; diagnostic imaging ; pathology ; Carcinoma, Hepatocellular ; therapy ; Chemoembolization, Therapeutic ; adverse effects ; Cholangiography ; Cisplatin ; administration & dosage ; Dilatation, Pathologic ; etiology ; Epirubicin ; administration & dosage ; Female ; Fluorouracil ; administration & dosage ; Follow-Up Studies ; Humans ; Iodized Oil ; administration & dosage ; Liver Neoplasms ; therapy ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Mitomycin ; administration & dosage ; Ultrasonography

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Cloning, Molecular ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; genetics