Aged ; Aged, 80 and over ; Airway Resistance ; Forced Expiratory Volume ; Humans ; Oscillometry ; Prospective Studies ; Pulmonary Disease, Chronic Obstructive ; diagnosis ; physiopathology ; Respiratory Function Tests ; methods ; Sensitivity and Specificity

OBJECTIVETo explore the effects of naloxone on the expression of c-kit receptor (c-kit R) and its ligand stem cell factor (SCF) in human embryo neuronal hypoxic injury.

METHODSSerum-free cerebral cortical cultures prepared from embryonic human brains were deprived of both oxygen and glucose which would set up an environment more likely with that of in vivo ischemic injury. Neurons in 24-well culture plates were randomly divided into four groups: control group, hypoxia group, naloxone 0.5 microg/ml group and naloxone 10 microg/ml group. MTT assay and biological analysis were performed to study the cell death and the changes of extracellular concentrations of lactate dehydrogenase (LDH) after combined oxygen-glucose deprivation. Neurons in 25 ml culture flasks were also randomly allocated into four groups as previously described. Intracellular total RNA were extracted at different time points: pre-hypoxia, immediately after hypoxia, and 3, 6, 12, and 24 hours after reoxygenation. The changes of SCF/c-kit R mRNA expression in hypoxic neurons treated with different concentrations of naloxone pre and post oxygen-glucose deprivation were determined with RT-PCR.

RESULTSThe cell vitality detected by MTT assay decreased significantly in hypoxia group and naloxone 0.5 microg/ml group when compared with control group (P<0.01), while no significant difference was found between naloxone 0.5 microg/ml group and hypoxia group or between naloxone 10 microg/ml group and control group. Extracellular concentration of LDH significantly increased in hypoxia group (P<0.05), while no difference was found between naloxone 0.5 microg/ml group and control group, between naloxone 0.5 microg/ml and hypoxia group, or between naloxone 10 microg/ml and control group (all P>0.05). Immediately after oxygen-glucose deprivation, the expression of SCF/c-kit R mRNA increased significantly (P<0.01). Among those the expression of SCF presented a distribution of double-peak value within 24 hours. After treated with different concentrations of naloxone, the peak value of each group were delayed to appear and went down with the increasing of naloxone concentration. The peak values in all treated groups were significantly different from that in control group (P<0.01).

CONCLUSIONSThe expression of SCF/c-kit R mRNA increases at the early stage after combined oxygen-glucose deprivation. Naloxone 0.5 microg/ml can attenuate cell injuries and regulate the expression of SCF/c-kit R. Naloxone may protect neurons by modulating the expressions of some cytokines.

Cell Hypoxia ; drug effects ; physiology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Humans ; Naloxone ; pharmacology ; Neurons ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics ; Stem Cell Factor ; genetics ; metabolism

OBJECTIVETo investigate the role of Caspase-8 and Bcl-2 in the formation of loose bodies in Kashin-Beck disease (KBD).

METHODSSpecimens of cartilage loose bodies were collected from 50 adult patients with KBD, and the samples of articular cartilage were collected from 10 healthy adults to serve as control. Avidin-biotin alkaline phosphatase immunohistochemistry was employed to examine Bcl-2 and Caspase-8 positivities in the chondrocytes in the loose bodies.

RESULTSIn KBD loose bodies, the percentage of chondrocytes positive for Bcl-2 and Caspase-8 [(18.40∓8.78)% and (67.54∓12.29)%, respectively] were significantly higher than those of the control group [(12.25∓1.58)% and (24.70∓4.35)%, respectively]. Caspase-8 was found to promote chondrocyte apoptosis in the loose bodies, and this effect overrode the apoptosis-suppressing effect of Bcl-2. Bcl-2 and Caspase-8 positivities were found mainly in the deep hypertrophic chondrocytes in the cartilage or in cells adjacent to the bone tissues.

CONCLUSIONKBD loose bodies contain an increased percentage of apoptotic chondrocytes positive for Bcl-2 and Caspase-8. The apoptosis-inducing effect of Caspase-8 was a dominant feature in the cartilage pathology of KBD compared to the apoptosis-suppressing effect of Bcl-2.

Adult ; Apoptosis ; Cartilage ; pathology ; Case-Control Studies ; Caspase 8 ; metabolism ; Female ; Humans ; Joint Loose Bodies ; metabolism ; Kashin-Beck Disease ; metabolism ; pathology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo explore the role of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) in myocardial ischemia/reperfusion injury (MI/RI) by observing the dynamic expression changes at mRNA and protein levels early after myocardial ischemia/reperfusion (I/ R).

METHODSThe Wistar rats were randomly divided into Sham and I/R group (n = 42), and killed according to different reperfusion time (1, 2, 4, 6, 12, 24 h and 7 d). Structural and morphous changes of myocytes were observed under optical microscope. The mRNA and protein levels of TLR2 and TLR4 were detected using real-time PCR (RT-PCR). Monocyte chemokine protein-1 (MCP-1) and interleukine-6 (IL-6) mRNA levels were measured by reverse transcriptase-polymerase chain reaction (rt-PCR).

RESULTS(1) With the extension of reperfusion time, the myocardial infarct size increased smoothly, and reached the plateau at 4 h, then stayed in the platform. After reperfusion for 7 d, the ventricular had been remodeled. (2) At the beginning of reperfusion, myocardial structure showed no significant change in Sham group, but had different degrees of injury in I/R group. In rats of the group reperfused for 7 d the left ventricular remodeling could be visible. (3) Compared to sham group,TIR2, TLR4, MCP-1, IL-6 mRNA level were increased in myocardium in I/R group. TLR2 and TLR4 both peaked at 4 h of reperfusion, IL6 peaked at 6 h, followed by a gradually decrease. TLR4 and IL-6 mRNA levels rose again at 7 d. MCP-1 level in I/R group remained fairly with sham group at the beginning of reperfusion, and markedly elevated at 7 d.

CONCLUSIONExpression of TLRs mRNA in myocardium during early after myocardial ischemia/reperfusion increased rapidly and activated TLRs might play an important role in MI/RI through promoting the generation of inflammatory factors. At the late reperfusion, TLRs levels raise again and the expression of inflammatory factors increase once again, Those may probably affect the remodeling of ventricular, and injure myocardial structure and function.

Animals ; Chemokine CCL2 ; metabolism ; Disease Models, Animal ; Interleukin-6 ; metabolism ; Male ; Myocardial Reperfusion Injury ; metabolism ; Rats ; Rats, Wistar ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.
Amino Acid Sequence ; Base Sequence ; Desulfurococcaceae ; enzymology ; genetics ; Endo-1,4-beta Xylanases ; genetics ; metabolism ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Hot Temperature ; Molecular Sequence Data ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics ; Structure-Activity Relationship

A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was made by Swiss-Model. The hydrophobic Interaction between beta-sheet B1 and B2 in the tertiary structure model of XYNB was compared with other thermophilic xylanase. A T11Y mutation was introduced in XYNB by site-dirrected mutagenesis to improve the thermostability of the enzyme. The XYNB and mutant xylanase (XYNB') expressed in Pichia pastoris were purified and their enzymatic properties were determined. The result revealed that the thermostability of XYNB' was obviously higher than that of XYNB. The optimal temperature of XYNB' for its activity was 60 degrees C, similar to XYNB. But, compare to XYNB, the optimal pH value, the Km value and the specific activity of XYNB' had also been changed. The research results suggested that the aromatic interaction between beta-sheet B1 and B2 in xylanase should increase enzyme thermostability. The mutant xylanase XYNB' is a good material for further research in the relationship between structure and function of xylanase.
Bacterial Proteins ; chemistry ; genetics ; Endo-1,4-beta Xylanases ; chemistry ; genetics ; Enzyme Stability ; Hot Temperature ; Hydrophobic and Hydrophilic Interactions ; Mutagenesis, Site-Directed ; Mutant Proteins ; chemistry ; Pichia ; genetics ; metabolism ; Protein Conformation ; Protein Folding ; Protein Structure, Tertiary ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces ; enzymology ; genetics ; beta-Glucosidase ; chemistry ; genetics

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.
6-Phytase ; genetics ; Fermentation ; Gene Dosage ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombination, Genetic

OBJECTIVETo investigate the short-term efficacy of complete mesocolic excision (CME).

METHODSClinical data of 62 cases of colon cancer (I-III phase) with radical resection including CME surgery group of 31 cases and traditional surgery group of 31 cases from January 2011 to October 2011 in Peking University People's Hospital were retrospective analyzed.

RESULTSThe number of removed lymph node in CME and traditional resection group was 22.5±1.8 and 17.6±1.3 respectively (P<0.05) and the positive rate of lymph node in mesentery root was 9.7% (3/31) in CME surgery group. Operative blood loss was (123.5±17.6) ml and (143.5±15.3) ml in CME and traditional resection group without significant difference (P>0.05). Except for more abdominal drainage volume of 3 days post-operation in CME group (P<0.05), the postoperative recovery indicators of postoperative drainage tube removed time, exhaust time, eating time, and the socioeconomic effects indicators of postoperative hospitalization, hospitalization costs were not significantly different between two groups (all P>0.05). Postoperative intestinal obstruction occurred in 3 cases and 4 cases, lymph fistula in 2 cases and 0 case, wound dehiscence in 1 case and 1 case in CME group and traditional resection group respectively. Postoperative complication rate was not significantly different (19.4% vs. 16.1%, P>0.05).

CONCLUSIONCompared with traditional radical surgery, CME sweeps lymph nodes more thoroughly, including lymph nodes of mesocolic roots, and does not affect postoperative recovery and increase the risk of postoperative complications.

Adult ; Aged ; Aged, 80 and over ; Colonic Neoplasms ; surgery ; Female ; Humans ; Lymph Node Excision ; Male ; Mesocolon ; surgery ; Middle Aged ; Retrospective Studies ; Treatment Outcome

Anti-Bacterial Agents ; pharmacology ; Bacterial Outer Membrane Proteins ; Bacterial Typing Techniques ; Electrophoresis, Gel, Pulsed-Field ; Microbial Sensitivity Tests ; Neisseria meningitidis, Serogroup B ; classification ; drug effects ; genetics ; Phylogeny