Objective Investigation of biological characteristics of Cdc 20AAA/+APCmin/+ mouse embryonic fibroblast(MEFs) indicate the effect of Cdc20AAA/+on growth of mouse embryonic fibroblast and the possible mechanism . Methods MEFs of Cdc20AAA/+APCmin/+, Cdc20AAA/+, APCmin/+ and WT genotype were harvested from embryos for analysis.The growth characteristics of Cdc20AAA/+APCmin/+, Cdc20AAA/+,APCmin/+and WT mouse embryonic fibroblast were analyzed through growth curve analysis and foci formation assay .Separation of sister chromatid and the presence of aneuploid were detected by karyotype analysis .Results Cell proliferation assays showed that Cdc 20AAA/+APCmin/+cells grew at an accelerated rate compared with APC min/+MEFs(P<0.01).Foci formation assay showed that the clone forming ability was significantly increased .Cdc20AAA/+APCmin/+MEFs showed a significant increase in the frequency of aneuploid compared with WT MEFs , which had a karyotype of 38 and contained prematurely separated sister chromatids .Conclusion Cdc20 carrying a null allele (Cdc20AAA/+) may accelerate the growth and proliferation of APC min/+MEFs and present the growth characteristics of the tumor cells .The possible mechanism may be associated with chromosome instability .

The cases of lymphoma accompanied or preceded by Coombs' test positive autoimmune hemolytic anemia (AIHA) have been reported. However, Coombs' test negative AIHA prior to the diagnosis of lymphoma was rarely described. Herein, this article reports a case of non-Hodgkin's lymphoma (NHL) preceded about 1.5 years by Coombs test negative AIHA. A woman aged 69 was diagnosed with HA based on the history and laboratory tests. Further studies revealed that this patient was negative with Coombs' test for IgG, IgM, IgA and C3. After all possible causes of HA, especially malignancies were ruled out, the patient was diagnosed with Coombs' test negative AIHA and treated with prednisolone. The patient responded well initially to steroid treatment. Two recurrences of acute HA were presented at time of 10 months post steroid cessation, and immediately after an attempt to withdraw steroid, respectively, but the hemolysis was effectively controlled by reinstitution of prednisolone. At third recurrence, however, the patient was no longer responding to steroid, and was found with cervical lymphadenopathy. Coombs' test for IgG, IgM, IgA and C3 remained negative. B cell NHL was diagnosed by pathology. After receiving 6 cycles of CHOP chemotherapy, the patient was lymphoma free, but the hemolysis was not improved, however, which was effectively controlled by the following low dose-rituximab (RTX) therapy. The patient was still kept in a remission of lymphoma free of anemia. In conclusion, this report presented a very rare case of NHL with Coombs' test negative AIHA as initial major clinical manifestation.
Aged ; Anemia, Hemolytic, Autoimmune ; diagnosis ; etiology ; therapy ; Antibodies, Monoclonal, Murine-Derived ; therapeutic use ; Coombs Test ; Female ; Humans ; Lymphoma, Non-Hodgkin ; complications ; diagnosis ; Rituximab

OBJECTIVETo study the growth inhibition effect of Brucea javanica Fruit Oil Emulsion (BJFOE) on human ovarian caner SKOV3 cells and the transplanted tumor of SKOV3 nude mice.

METHODSGrowth inhibition effects of different concentrations BJFOE alone or its combination with cisplatin on human ovarian cancer cell SKOV3 were measured using MTT method. The orthotopic transplantation tumor model of human ovarian cancer SKOV3 cell lines was established in nude mice. Totally 32 ovarian cancer nude mice were randomly divided into 4 groups, i.e., the blank control group (Group A), the BJFOE group (Group B), the BJFOE combined Cisplatin group (Group C), and the Cisplatin control group (Group D), 8 in each group. Mice in Group A were intraperitoneally injected with normal saline (0.2 mL/ 20 g), once per two days. Mice in Group B were intraperitoneally injected with BJFOE (0.2 mL/20 g), once per two days. Mice in Group C were intraperitoneally injected with cisplatin (3 mg/kg) 0.2 mL on the first day, and intraperitoneally injected with BJFOE on the second day. Mice in Group D were intraperitoneally injected with cisplatin (3 mg/kg) 0.2 mL, once per two days. All mice were injected for six times, and sacrificed 48 h after the last injection. The lesion formation of the abdominal tumor tissue was observed. Tumor specimens were obtained to perform HE staining. Expression levels of MRP-1/CD9 and integrinα-5 were detected using Western blot.

RESULTSThe inhibition of BJFOE was time-dose depend- ently correlated with its inhibition effect of SKOV3 cells. The inhibition effect of BJFOE in combination of cisplatin was significantly superior to that of using any of the two drugs alone. Western blot results showed expression levels of MRP-1/CD9 and integrinα-5 were up-regulated in Group B and Group D with statistical difference (P < 0.05). But they were down-regulated in Group C with statistical difference (P < 0.05).

CONCLUSIONSIntraperitoneal injecting BJFOE was feasible and effective for treating ovarian cancer. BJFOE also could inhibit the invasion and migration of tumor cells targeting at MRP-1/CD9 and integrinα-5. But its specific anti-tumor mechanism was not clearly probed.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Brucea ; Cell Line, Tumor ; Cisplatin ; Female ; Fruit ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Ovarian Neoplasms ; Plant Oils ; pharmacology

OBJECTIVETo observe the influence of lead exposure on the activity of nitric oxide synthase (NOS) in different brain regions of rat.

METHODSBy establishing a series of rat models exposed to different low levels of lead (drinking water containing 0.025%, 0.050%, 0.075% of lead acetate) during developing period, NOS activities in hippocampus, cerebellum, cerebral cortex and brain stem were studied.

RESULTSOn the 21st day after birth, NOS activities in hippocampus of three levels of lead exposed groups [(1.53 +/- 0.20), (1.66 +/- 0.23), (1.88 +/- 0.32) U/mg pro respectively], and in cerebellum [(0.87 +/- 0.24), (0.85 +/- 0.09), (0.91 +/- 0.18) U/mg pro respectively] were significantly lower than those of control group [(2.36 +/- 0.18), (1.41 +/- 0.18) U/mg pro, respectively, P < 0.01]. NOS activities in cerebral cortex of 0.075% group [at 7, 14, 21 d of age [(1.29 +/- 0.14), (1.03 +/- 0.15), (0.69 +/- 0.10) U/mg pro] were significantly lower than those in control group [(2.54 +/- 0.31), (1.64 +/- 0.22), (1.24 +/- 0.14) U/mg pro respectively], and 0.025% group [(2.42 +/- 0.19), (1.59 +/- 0.17), (1.27 +/- 0.12) U/mg pro respectively], and 0.050% group [(2.56 +/- 0.53), (1.77 +/- 0.19), (1.24 +/- 0.10) U/mg pro respectively, P < 0.05]. There were no significant differences among control, 0.025%, and 0.050% groups (P > 0.05). Lead exposure had no influence on NOS activity in brain stem at the same age (P > 0.05).

CONCLUSIONNOS activities in hippocampus, cerebellum and cerebral cortex were inhibited by low level lead exposure and the degree of the effect was related to Pb exposure time and/or level of Pb exposed.

Animals ; Brain ; drug effects ; enzymology ; Brain Stem ; drug effects ; enzymology ; Cerebellum ; drug effects ; enzymology ; Cerebral Cortex ; drug effects ; enzymology ; Dose-Response Relationship, Drug ; Female ; Hippocampus ; drug effects ; enzymology ; Lead ; toxicity ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors

The objective of this study was to investigate the prognosticating value of multiparameter flow cytometry in detection of minimal residual disease (MRD) and relapse risk of patients with acute myeloid leukemia (AML). Multiparameter flow cytometry (MPFC) analysis was used to detect the leukemia-associated aberrant immunophenotype (LAIP) of the pretreated patients with AML and to assess the levels of MRD after remission induction (Post-Ind MRD) and consolidation therapy (Post-Cons MRD). The results showed that the definite LAIP could be detected in 94.3% of the patients (115/122) with AML (except APL). Among 115 cases only one LAIP was identified in 15 cases (13.0%), but two or more LAIP were identified in other 100 cases (87.0%). The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0%and 34.8% respectively. MRD frequency was monitored in 41 AML patients with CR after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD(+) and 17 patients were Post-Ind MRD(-). The percentages of relapse in cases of Post-Ind MRD(+) and Post-Ind MRD(-) were 75.0% (18/24) and 29.4% (5/17) respectively after consolidation chemotherapy. The relapse free survival (RFS) times of the patients with Post-Ind MRD(+) and Post-Ind MRD(-) were 49.06 +/- 6.53 months and 11.92 +/- 1.64 months (p < 0.0001) respectively. 18 patients were Post-Cons MRD(+) and 23 patients were Post-Cons MRD(-). The percentages of relapse in cases of Post-Cons MRD(+) and Post-Cons MRD(-) patients were 100% (18/18) and 21.7% (5/23) respectively after consolidation chemotherapy. The RFS times of the patients with Post-Cons MRD(+) and Post-Cons MRD(-) were 41.74 +/- 5.52 months and 10.06 +/- 1.72 months (p < 0.0001) respectively. It is concluded that the levels of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. The detection of MRD by MPFC provides prognostic information in AML patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Flow Cytometry ; methods ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; pathology ; Male ; Middle Aged ; Neoplasm, Residual ; diagnosis ; pathology ; Prognosis ; Recurrence ; Young Adult

Microbial transformation of cardamonin by Mucor spinosus (CGMCC 3.3450) in preparative scale resulted in the isolation of two new products. Their structures were elucidated unambiguously by ESI-MS, 1H NMR, 13C NMR and 2D NMR spectra analyses as 4-O-beta-D-glucopyranosyl-6-hydroxy-2-methoxychalcone (1, 4-GluC) and 6-O-beta-D-glucopyranosyl-4-hydroxy-2-methoxychalcone (2, 6-GluC), respectively. The time-course of biotransformation by M. spinosus showed that both 4-GluC and 6-GluC appeared on the 2nd day. The optimal biotransformation temperature was 28 degrees C, the optimal biotransformation time was 72 h and the optimal concentration for cardamonin was 40 mg x mL(-1). This is the first time for successful microbial glycosylation of cardamonin in present research.
Biotransformation ; Chalcones ; chemistry ; isolation & purification ; metabolism ; Glucosides ; chemistry ; isolation & purification ; Glycosylation ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Mucor ; metabolism ; Spectrometry, Mass, Electrospray Ionization

This study was aimed to investigate the characteristics of immunophenotypes in the patients with myelodysplastic syndrome (MDS) without an increase of marrow blasts, and to confirm their diagnostic significance. Marrow cells from 222 patients with pancytopenia, dysplastic changes in one or more hematopoietic lineages and blast cells less than 5% were analyzed by multiparametric flow cytometry(FCM). The abnormal immunophenotypes were evaluated in asynchronous antigen expression (CD34 or CD117 in mature granulocytes or mature monocytes, HLA-DR in mature granulocytes), in cross-lineage antigen expression (CD7 or CD56 in granulocytes or monocytes), in aberrant light-scatter (CD45/SSC in mature granulocyte or monocyte) and in abnormal expression of differentiation antigen (CD13/CD16 pattern in granulocytes and HLA-DR under-expression in monocytes). The sensitivity and specificity of abnormal immunophenotypes were determined on diagnosis. Among 222 cases, 127 cases were diagnosed as MDS by traditional diagnostic method and 95 cases were non-MDS (drug-related neutropenia, autoimmune cytopenia and idiopathic thrombocytopenia). In mature granulocyte gate, the sensitivity of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC and abnormal expression of differentiation antigen were 31.5%, 30.7%, 49.6% and 60.6% respectively, and the specificity were 100%, 100%, 88.4% and 52.6% respectively. In monocyte gate, the sensitivity of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC and abnormal expression of differentiation antigen were 2.3%, 11%, 37% and 12.6% respectively. The specificity was 100% in all of them. Among 8 above mentioned items, sensitivity of more than 2 abnormalities was 77.9%, and specificity was 95.8%. The positive predictive value was 96.1%. It is concluded that the abnormal expression of asynchronous, cross-lineage antigen expression, aberrant light-scatter of CD45/SSC have a high specificity and a low sensitivity for diagnosis of MDS. The abnormal expressions of differentiation antigens have a high sensitivity and a low specificity; however, the detection of multiple expression abnormalities possesses the high sensitivity and specificity for diagnosis of MDS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; immunology ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; diagnosis ; immunology ; Sensitivity and Specificity ; Young Adult

To figure out the stability and intestinal bacteria metabolites of rats in vitro of astragaloside IV ( AST), this research was done to explore the stability of AST in the artificial gastric juice. artificial intestinal juice and rat liver homogenate and the metabolism in rat intestinal in vitro. HPLC was used to calculate the remaining rate of AST in biological samples by measuring the content of AST, while metabolites were determined by combining the methods of TLC, HPLC and LC-MS/MS. It turned out that AST was difficult to metabolize in the artificial gastric juice, artificial intestinal juice and rat liver. Also, the metabolic pathway of AST was stepped by deglycosylation. Firstly, AST was converted to its secondary etabolites (6-O-β-D-glucopyranosyl- cycloastragenol, CMG) by removal of xylose moiety at C-3, then transformed into cycloastragenol (CAG) after hydrolytic removal of the glucose moiety at C-6. All the results suggested that the metabolism of AST in vivo occurs mainly in the intestinal by hydrolysis of glycosyl. In conclusion, hydrolysis of intestinal flora is the main reason that AST metabolizes.
Animals ; Bacteria ; metabolism ; Chromatography, High Pressure Liquid ; Drug Stability ; Intestines ; microbiology ; Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Saponins ; chemistry ; metabolism ; Tandem Mass Spectrometry ; Triterpenes ; chemistry ; metabolism

OBJECTIVETo investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.

METHODSTotal RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively.

RESULTSPDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.

CONCLUSIONSThe construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Animals ; Cats ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Corneal ; cytology ; drug effects ; Humans ; Immunohistochemistry ; Phosphopyruvate Hydratase ; analysis ; Protein Folding ; Proto-Oncogene Proteins c-sis ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Wound Healing ; drug effects

OBJECTIVETo observe the proliferation state of transplanted cells in acute myocardial infarction (AMI) rats, and the endothelial progenitor cells (EPCs) preconditioned by salvianolic acid B in different ratios with the bone mesenchymal stem cells (BMSCs).

METHODSThe cultivation and purification of EPCs were performed by density-gradient centrifugation and plastic adherence method. Two types of cells were identified by immunocytochemical method (CD34, CD133, and CD44). The rat model of AMI was prepared by ligation of left anterior descending artery. The EPCs were pre-treated with the optimal concentration of salvianolic acid B (8 microg/ mL). They were mixed with BMSCs in different proportions (EPCs/BMSCs in the ratio of 1:1, 2:1, 4:1, and 8:1, respectively). BMSCs and EPCs were injected into the myocardial infarction area. The infarcted area was determined by the N-BT staining and hematoxylin-eosin staining. The expression of Ki-67 was detected by immunohistochemical assay.

RESULTSCompared with the model group (19.60% +/- 3.23%), the myocardial infarction area of each implanted group obviously decreased (P < 0.05). Of them, the decrease was most obvious in the 4:1 group (11.37% +/- 2.18%) and the 8:1 group (9.23% +/- 2.35%, P < 0.05). Compared with the model group (cell/high magnification, 5.17 +/- 2.31), the Ki-67 positive cell number of each implanted groups significantly increased (P < 0.05). Of them, the Ki-67 positive cell number was obviously higher in the 8:1 group (15.00 +/- 3.16, P < 0.05).

CONCLUSIONSEPCs pretreated by salvianolic acid B combined with BMSCs could reduce the myocardial infarcted area, improve the proliferation of BMSCs in the peripheral infarction and local ischemia. Besides, along with the increase of the implant proportion of EPCs, the infarct area was gradually reduced, and the proliferative expression was gradually enhanced.

Animals ; Benzofurans ; pharmacology ; Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; Endothelial Cells ; cytology ; drug effects ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocardial Infarction ; metabolism ; pathology ; Rats ; Rats, Wistar ; Transplantation Conditioning