OBJECTIVETo study the chemical constituents of Phellinus igniarius.

METHODCompounds were isolated by nomal phasc sillica gel and Al2O3 chromatography and reverse phase HPLC. Their structures were elucidated by spectroscopic methods including IR, MS and NMR.

RESULTFive flavonoids and two coumarins were isolated from Phellinus igniarius and their structures were identified as naringenin, sakuranetin, aromadendrin, folerogenin, eriodictyol, coumarin and scopoletin.

CONCLUSIONAll these compounds were obtained from this genus for the first time.

Flavanones ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Polyporaceae ; chemistry ; Scopoletin ; chemistry ; isolation & purification

OBJECTIVETo investigate the signal patterns of dual color dual fusion (DCDF) probe and extra signal (ES) probe in the detection of BCR/ABL fusion gene, and illustrate the relation between the fluorescence in situ hybridization (FISH) pattern and the karyotype.

METHODSSixty-five cases of chronic myelocytic leukemia (CML) and 50 cases of acute lymphoblastic leukemia (ALL) were detected by FISH with DCDF probe, the BCR/ABL positive samples were detected by FISH with ES probe. Among these cases, 47 cases of CML and 40 cases of ALL perform conventional cytogenetics simultaneously.

RESULTSAll 65 cases of CML were all BCR/ABL positive by FISH. 17 cases showed the atypical pattern by DCDF-FISH, and 12 cases showed the atypical pattern by ES-FISH. There were 7 cases of BCR/ABL positive in 50 cases of ALL by FISH. By ES-FISH, there were 5 cases in which the break-point of BCR gene was located in m-bcr, 2 cases in which the break-point of BCR gene was located in M-bcr. Conventional cytogenetics demonstrated that 43/44(98%) cases of CML and 7/32(22%) cases of ALL were Ph positive.

CONCLUSIONThe features of DCDF-FISH, ES-FISH and conventional cytogenetic are different from each other. According to the features of these method, it can increase the precision of the adjustment of genetic feature to analyze these results comprehensively.

Adult ; Cytogenetics ; methods ; DNA Probes ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; methods ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

BACKGROUNDThe genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.

METHODSThe prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.

RESULTSWe expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.

CONCLUSIONThe results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

Amino Acid Sequence ; Animals ; BALB 3T3 Cells ; Cercopithecus aethiops ; Growth Inhibitors ; analysis ; physiology ; HeLa Cells ; Humans ; Immunohistochemistry ; Lung ; chemistry ; Mice ; Molecular Sequence Data ; SARS Virus ; chemistry ; Severe Acute Respiratory Syndrome ; metabolism ; Vero Cells ; Viral Structural Proteins ; analysis ; physiology

OBJECTIVETo establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.

METHODSOsteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively.

RESULTSThe co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change.

CONCLUSIONSCo-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.

3T3 Cells ; Animals ; Cell Differentiation ; Coculture Techniques ; Mice ; NF-kappa B ; metabolism ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; RAW 264.7 Cells ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore the value of CD3CD4T cell count in prediction of viral infections after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in the patients with severe aplastic anemia(SAA).

METHODSA total of 78 SAA patients with allo-HSCT in Guangzhou First People's Hospital from January 2014 to July 2016 were enrolled in this study. The absolute numbers of CD3CD4T cells were measured by flow cytometry at 1,2,3,6, and 12 month after allo-HSCT. According to the cell counts, the patients were divided into 3 groups: i.e. <50/µl (n=120), 50-100/µl(n=48) and >100/µl(n=123)groups. The infection incidences of human cytomegalovirus (HCMV) and Epstein-Barr virus(EBV) within 2 weeks around each time point were compared between different groups. According the counts of CD3CD4T cells at 3 months after-transplant, these patients were divided into 2 groups, i.e.>100/µl (n=30) and ≤100/µl (n=48). The incidences and duration of HCMV and EBV infection, overall survival rate were compared between 2 groups.

RESULTSThe incidences of CMV and EBV infection significantly decreased in CD3CD4T cell >100/µl group as compared with <50/µl and 50-100/µl groups. At 3 months after-transplant, there was lower incidence rates of CMV disease, EBV infection, shorter durations of CMV infection and better survival in CD3CD4T cell >100/µl group as compared with ≤100/µl group.

CONCLUSIONCD3CD4T cell count is a good predictor for CMV and EBV infection after allo-HSCT in SAA patients. There are low risk of infe-ctions from CMV and EBV when CD3CD4T cell count >100/µl in any time after transplant, which means lower occurrence of CMV and EBV infection and better survival when CD3CD4T cell counts is >100/µl in 3 months after transplant in SAA patients.