Chaetomium thermophilum CT2 can produce extracellular cellulase with industrial value. We designed two degenerate primers to amplify catalytic domain sequence of cellobiohydrolase II ( CBH II). Full length of cDNA was obtained by rapid amplification of cDNA ends technologies. DNA sequencing revealed that cbh2 has an open reading frame of 1428bp, which encodes a putative polypeptide of 476 amino acids. The deduced amino acid sequence shows that the predicted molecular mass is 53 kD and the cbh2 consists of a fungal-type carbohydrate binding domain (CBD) separated from a catalytic domain by a linker region rich in proline/serine/threonine. PCR product consisting of the entire CBH II coding region without its signal sequences was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS115. Highly efficient production of the cellobiohydrolase II was achieved in P. pastoris under the control of the AOX1 promoter, and the expressing level was 1.2 mg/mL by small-scale culturing. The recombinant cellobiohydrolase II was purified by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography. A molecular mass of the purified enzyme is 67 kD determined by SDS-PAGE and this is similar to the native cellobiohydrolase II purified from C. thermophilum CT2. The recombinant enzyme exhibited optimum catalytic activity at pH 4.0 and 50 degrees C respectively. It was thermostable at 50 degrees C and retained 50% of its original activity after 30 min at 70 d degrees C . The high level of fully active recombinant cellobiohydrolase II got from P. pastoris makes this expression system attractive for fermentor and industrial applications.
Amino Acid Sequence ; Base Sequence ; Cellulose 1,4-beta-Cellobiosidase ; biosynthesis ; genetics ; Chaetomium ; enzymology ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo evaluate the differences in the autophagy activity of alveolar macrophages between patients with different stages of coal workers' pneumoconiosis (CWP).

METHODSA total of 116 coal workers were investigated in the field. Their lung lavage fluid was collected and purified to obtain alveolar macrophages. The morphological characteristics of autophagy were observed by transmission electron microscopy. The expression of autophagy marker (LC3) and autophagy regulators (Beclin1, mTOR, and p-mTOR) was measured by Western blot. The autophagy activity of alveolar macrophages was compared between dust-exposed subjects and patients with stage I, II, and III CWP.

RESULTSThe autophagy activity of alveolar macrophages differed between patients with different stages of CWP, according to transmission electron microscopy. Patients with stage II CWP had significantly higher protein expression of LC3 II/I and Beclin1 in pulmonary macrophages than those with stage ICWP (P < 0.05); patients with stage III CWP had significantly lower protein expression of LC3 II/I and Beclin1 in pulmonary macrophages than those with stage II CWP (P < 0.05), but had significantly higher protein expression of LC3 II/I and Beclin1 than those with stage I CWP (P < 0.05); patients with stage II CWP had a significantly higher protein expression of Beclin1 than the dust-exposed subjects (P < 0.05). Patients with stage II CWP had significantly lower expression of mTOR and p-mTOR in pulmonary macrophages than the dust-exposed subjects and those with stage I CWP (P < 0.05), while patients with stage III CWP had significantly higher expression of mTOR and p-mTOR than those with stage II CWP (P < 0.05).

CONCLUSIONThe autophagy activity of alveolar macrophages varies between patients with different stages of CWP.

Anthracosis ; pathology ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Biomarkers ; Bronchoalveolar Lavage Fluid ; Coal ; Coal Mining ; Dust ; Humans ; Macrophages, Alveolar ; pathology ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Occupational Exposure ; Pneumoconiosis ; pathology

OBJECTIVETo screen the genes related with leukocyte responses in mice early after burn injury by bioinformatic analysis of the gene expression profiling data.

METHODSGene expression profiles were obtained from GEO (GSE7404, Mouse musculus, 25% TBSA, full-thickness) database. After screening of the differentially expressed genes (DEGs) through paired-sample t-test and fold-change, DAVID online tools were used to select the DEGs related to leukocyte responses to burns by GO functional enrichment analysis; the interacting genes identified through KEGG pathway enrichment analysis were transferred to STRING to construct the protein-protein interaction (PPI) network. Biological annotation of the sub-networks was executed using the software Cytoscape. Real-time PCR was used to verify the DEGs identified in mice.

RESULTSOf the 259 leukocyte response-related DEGs screened at 1 day post-burn, 118 were up-regulated and 141 were down-regulated. KEGG pathway enrichment analysis showed that the pathways were associated with the immune function, cell growth and cell death. PPI network and module analysis suggested that some of genes (such as Lck, Stat1, Myd88, Stat3, and Jun) play critical roles in the PPI network post-burn. RT-PCR results were consistent with those of bioinformatic analysis.

CONCLUSIONSLck, Stat1, Myd88, Stat3, and Jun might be critical players in the development of leukocyte response in mice early after burn injury. Our finding provides new insights into the pathogenesis of leukocyte response to burn injury and identifies several potential biomarkers for burn treatment.

Animals ; Burns ; genetics ; Computational Biology ; Down-Regulation ; Gene Expression Profiling ; Gene Regulatory Networks ; Leukocytes ; physiology ; Mice ; Real-Time Polymerase Chain Reaction ; Software ; Up-Regulation

OBJECTIVEThe explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.

METHODSThirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.

RESULTSThe half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.

CONCLUSIONIn the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.

Animals ; Calcium ; metabolism ; Cecum ; Diaphragm ; drug effects ; metabolism ; Endoplasmic Reticulum ; metabolism ; Ligation ; Male ; Muscle Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sepsis

OBJECTIVETo investigate the effects of treadmill exercise on central hemodynamics in patients with coronary artery disease.

METHODSSixty-eight consecutive patients below 60 years of age with the diagnosis of coronary artery disease (CAD) between July, 2013 and April, 2014 underwent treadmill exercise test following the standard Bruce protocol. Ninety-seven individuals without CAD served as the control group. Central hemodynamics of the subjects, including the central aortic blood pressure (cSBP), augmentation index (AI) and augmentation pressure (AP), were examined before and after the exercise.

RESULTScSBP increased significantly after acute exercise in both groups (P<0.001). Immediately after treadmill exercise, AI showed no significant changes in CAD patients (P=0.561) but decreased significantly in the control subjects (P<0.001). AI before exercise and cSBP after exercise were significantly higher in CAD group than in the control group (P=0.009 and 0.009, respectively). Stepwise regression analysis showed that the maximal heart rate after exercise was the main factor that affected cSBP increment in CAD group (P=0.012), and the occurrence of ischemia after exercise was associated with a lower cSBP increment (P=0.048).

CONCLUSIONAI does not decrease significantly after acute exercise in patients with CAD, suggesting that AI is closely associated with coronary artery blood perfusion after exercise and may serve as a potential target for improving ischemic threshold during rehabilitation of the patients.

Blood Pressure ; Case-Control Studies ; Coronary Artery Disease ; physiopathology ; Exercise Test ; Heart Rate ; Hemodynamics ; Humans ; Middle Aged

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo study the immunophenotype and gene rearrangement pattern of pulmonary lymphomatoid granulomatosis.

METHODSNine cases of pulmonary lymphomatoid granulomatosis, included 5 cases of open lung biopsy, 3 cases of lobectomy specimen and 1 case of autopsy, were retrospectively analyzed by immunohistochemistry, in-situ hybridization for Epstein-Barr virus-encoded RNA, immunoglobulin and T-cell receptor gene rearrangement studies.

RESULTSThe age of patients ranged from 3 to 59 years. The male-to-female ratio was 3: 6. Histologically, all cases showed lymphocytic infiltration surrounding the blood vessels and in the perivascular areas. Most of these lymphoid cells expressed T-cell marker CD3. There were also variable numbers of CD20-positive B cells. The staining for CD56 was negative. According to the WHO classification, there were 4 cases of grade I , 1 case of grade II and 4 cases of grade III lesions. Six cases had gene rearrangement studies performed and 3 of them demonstrated clonal immunoglobulin gene rearrangement (including 1 of the grade II and 2 of the grade III lesions). No T-cell receptor gene rearrangement was detected.

CONCLUSIONSPulmonary lymphomatoid granulomatosis may represent a heterogeneous group of lymphoproliferative disorders. Some of the cases show B-cell immunophenotype and clonal immunoglobulin gene rearrangement, especially the grade II and grade lesions. They are likely of lymphomatous nature.

Adult ; Antigens, CD20 ; metabolism ; CD3 Complex ; metabolism ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Humans ; Immunohistochemistry ; Lung Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Lymphomatoid Granulomatosis ; genetics ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Neoplasm Grading ; Pneumonectomy ; methods ; Retrospective Studies ; Young Adult

OBJECTIVETo explore the methods for constructing the digital three-dimensional model of fetal heart.

METHODSOriginal two-dimensional CT image data sets were collected from 4 abortion fetuses with fetal malformations but not heart malformation or chromosomal abnormalities. The three-dimensional fetal heart model was reconstructed using Mimics14.0 software.

RESULTSIn the reconstructed three-dimensional fetal heart, the left atrium, left ventricle, right atrium, right ventricle, the ascending aorta, the main pulmonary and their branches, the superior cava and inferior vena cava were marked with different colors, and these structures could be displayed individually or with other structures. This model also allowed three-dimensional arbitrary scaling, shifting or rotation at any angle, and the diameter of the each vessel could be measured with the software.

CONCLUSIONThe fetal heart model can be successfully reconstructed from the CT datasets using three-dimensional reconstruction software to facilitate clinical and anatomical teaching.

Female ; Fetal Heart ; anatomy & histology ; Heart Atria ; anatomy & histology ; Heart Defects, Congenital ; Heart Ventricles ; anatomy & histology ; Humans ; Imaging, Three-Dimensional ; Models, Anatomic ; Pregnancy ; Software ; Tomography, X-Ray Computed ; Vena Cava, Inferior ; anatomy & histology

To cultivate CD34(+)CD38(-) cells isolated from umbilical cord blood of healthy puerperal women over a longer-period of time for observation of cell division, proliferation, apoptosis, and effects of stem cell factor on the growth of CD34(+)CD38(-) cells, with flow cytometry, CD34(+)CD38(-) cells were isolated from umbilical cord blood of 10 healthy puerperal women and cultivated in stem cell media with supplement of IL-3, IL-6, GM-CSF, EPO, IGF-1 and SCF 6 kinds cell growth stimulating factors for six months. The cell growth curves were established. The effects of stem cell factor on the growth of CD34(+)CD38(-) cells and cell apoptosis were investigated with the single cell gel electrophoresis technique and flow cytometry method, respectively. The results showed that CD34(+)CD38(-) cells isolated from umbilical cord blood were capable of proliferating after being cultivated in vitro over a longer-period of time with no evidence of the presence of excessive apoptosis. In conclusion, under appropriate culture conditions, CD34(+)CD38(-) hematopoietic early progenitor cells from umbilical cord blood can serve as a resource providing a large amount of primitive cells for transplantation therapy after a longer period of cultivation and proliferation in vitro.
ADP-ribosyl Cyclase 1 ; analysis ; Adult ; Antigens, CD34 ; analysis ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans

OBJECTIVETo screen the genes related with leukocyte responses in mice early after burn injury by bioinformatic analysis of the gene expression profiling data.

METHODSThe gene expression profiles were obtained from GEO (GSE7404, Mouse musculus, 25% TBSA, full-thickness) database. T test, fold changes and GO functional enrichment analysis were used to identify the differentially expressed genes (DEGs) related to leukocyte responses to burns; the interacting genes were transferred to STRING to construct the protein-protein interaction (PPI) network. Biological annotation of the sub-networks was executed using the software Cytoscape. Real-time PCR and Western blotting were used to verify the DEGs in mice.

RESULTSIn mice at 1 day post-burn, a total of 658 genes were up-regulated and 1167 were down-regulated. PPI network and module analysis suggested that some of the genes (Stat1, Cdk1, Cd19, Lck and Jun) may play critical roles in the PPI network post-burn. Real-time PCR and Western blotting results in mice were consistent with those of bioinformatic analysis of Stat1, Cdk1 and Jun.

CONCLUSIONStat1, Cdk1 and Jun might be critical players in the development of leukocyte response in mice early after burn injury. Our finding provides new insights into the pathogenesis of leukocyte response to burn injury and identifies several biomarkers as potential targets for burn treatment.