A method for the determination of methylmercury in seafood has been developed using dispersive liquid-liquid microextraction followed by direct mercury analyzer. Total mercury was detected by direct mercury analyzer, and inorganic mercury was calculated by the difference. The parameters affecting the extraction efficiency, including the selection of extractant and dispersant, their volume ratio, concentration of HCl and NaCl have been optimized in this study. The results showed that CH2 Cl2 as extractant, ethanol as dispersant, Volume ration of 1:5, 1 mol/L HCl and 120 g/L NaCl were chosen. The detection limit and the dynamic liner range were 0. 10 μg/L and 0. 2-20 μg/L, respectively. The relative standard deviation was 6. 0% for eleven replicates at the spiked level of 2. 0 μg/L. The enrichment factor was 8. For total Hg determination, the detection limit and the dynamic liner range for methylmercury were 0. 10 μg/kg and 0. 2-50 μg/kg, respectively. The relative standard deviation was 2. 4%. The method was simple, fast and a little solvent needed. Some certified reference materials were analyzed to validate the accuracy of the proposed method, and the results were in good agreement with the reference value. Besides, the method was applied to the real samples with satisfactory results.

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.
Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; CD13 Antigens ; antagonists & inhibitors ; metabolism ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cell Line, Tumor ; Humans ; Leucine ; analogs & derivatives ; pharmacology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Phosphorylation ; Tretinoin ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the role of MHC class II Transactivator (C II TA) in expression of HLA molecules in five human malignant hematological cell lines.

METHODSThe expressions of HLA molecules and C II TA protein were detected by immunohistochemistry and flow cytometry. The expression of C II TA gene was detected by RT-PCR. The response of peripheral T cells after stimulation by Jurkat cells was detected by mixed lymphocyte reaction.

RESULTThe HLA II-positive tumor cells expressed the C II TA and IFN-gamma induced the expression of HLA I, II in tumor cells, which were able to express C II TA constitutively.

CONCLUSIONThere is a correlation between the inability of the tumor cells in response to IFN-gamma for HLA expression and the deficiency in the inducible expression of C II TA.

Cell Line, Tumor ; Genes, MHC Class II ; genetics ; HL-60 Cells ; HLA Antigens ; biosynthesis ; genetics ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology ; Nuclear Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; U937 Cells

OBJECTIVETo investigate the effects of soluble M-CSF receptor (sMR) on proliferation and differentiation of hematopoietic precursors derived from umbilical cord blood in mesenchymal stem cell (MSC) microenvironment.

METHODIn group of cytokine (CK) + sMR, MSCs were used as feeder cells, mononuclear cells (MNCs) from cord blood were expanded in MSC microenvironment in presence of SCF, Flt3L, TPO, IL-6 and sMR. In CK control group, no sMR was added. MNC counting and colony forming cell (CFC) culture were performed at week 1, 2, 3 and 4.

RESULTS1) The number of MNCs increased rapidly in both group CK and group CK + sMR (108.47 -fold and 120.67 -fold, respectively, P > 0.05). 2) CFC increased, peaked at week 3(38.1 x 10(3)) and declined rapidly at week 4(18.1 x 10(3)) in group CK, but still increased in group CK + sMR at week 4 (84 x 10(3)), the total number of CFC was higher in group CK + sMR than in group CK at week 3 and week 4 (P <0.01). 3) The erythroid CFC peaked at week 1 (5891.2 and 5635.6 for groups CK and CK + sMR, respectively), then dropped rapidly and to zero at week 3, in both group CK and group CK + sMR (P > 0. 05). 4) Myeloid CFC expanded continuously and peaked at week 3 (31.5 x 10(3)), then declined at week 4 (18.3 x 10(3)) in group CK; but still increased at week 4(80.8 x 10(3)) in group CK + sMR, being higher than that in group CK at week 3 and week 4 (P <0.01).

CONCLUSIONsMR can inhibit the differentiation of cord blood hematopoietic precursors expanded in MSC microenvironment, but the inhibition exerts only on myelomonocytic but not on erythroid precursors.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Mesenchymal Stromal Cells ; Receptor, Macrophage Colony-Stimulating Factor ; chemistry

OBJECTIVETo investigate the effects of interleukin 7 (IL-7) on B7 molecules expression and immunogenicity of acute leukemia (AL) cells.

METHODSThe B7 molecules expression on fresh acute leukemia cells and on the IL-7 exposed leukemia cells was detected by FACScan cytometer. B7-1 and B7-2 mRNA in IL-7 treated HL-60 cells were detected by reverse transcription-PCR (RT-PCR). The stimulation of proliferation of allogeneic peripheral blood mononuclear cells (PBMNC) by IL-7 treated leukemia cells was detected by MTT method. The level of interferon-gamma (IFN-gamma) secreted by the stimulated PBMNC was determined using enzyme-linked immunosorbent assays (ELISA). The blocking experiments were performed using monoclonal antibodies against B7-1, B7-2 and W6/32.

RESULTSB7-1 was weakly expressed in three, whereas B7-2 did in only one of eleven AL patients. IL-7 significantly enhanced B7 molecules expression on AL cells in a time-dependent manner. Furthermore, IL-7 could induce higher expression of B7-1 and B7-2 mRNAs on HL-60 cells. IL-7 treated leukemia cells could stimulate PBMNC proliferation and promote their IFN-gamma production. Anti-B7-1 and anti W6/32 but not anti-B7-2 monoclonal antibodies significantly inhibited the stimulated PBMNC proliferation and IFN-gamma secretion.

CONCLUSIONFresh AL cells express low level of B7-1 and B7-2 molecules. IL-7 enhances the B7 molecules expression on AL cells. The IL-7-treated leukemia cells can significantly stimulate the proliferation of allogeneic PBMNC and induce their IFN gamma secretion.

B7-1 Antigen ; genetics ; metabolism ; B7-2 Antigen ; genetics ; metabolism ; Humans ; Interleukin-7 ; pharmacology ; Leukemia ; immunology ; metabolism ; RNA, Messenger ; genetics ; Tumor Cells, Cultured ; Up-Regulation ; drug effects

PurposeTo investigate the clinical value of inverted X-ray plain film and MRI examination in the diagnosis of congenital anorectal malformation (CARM). Materials and Methods Thirty-eight cases with operatively proved anorectal malformation were reviewed; inverted X-ray plain film and MRI examination were performed in all patients before surgery. The relationship between the rectum blind side and pubococcygeal line (PC line), and the type of anal atresia was determined, to compare the diagnostic accuracy of inverted plain film with MRI for CARM typing. Results Of all the 38 cases, 19 cases were with low imperforate anus, 8 cases with median imperforate anus, and 11 cases with high imperforate anus. The accuracy rate of inverted X-ray plain film and MRI examination for the diagnosis of CARM typing was 92.1% (35/38) and 97.4% (37/38) respectively, and the difference between them was not statistically significant (χ2=1.37, P>0.05). 7 cases of fistula, 5 cases of spinal cord malformations and 1 case of right kidney agenesis can be clearly demonstrated on MRI. Conclusion Both inverted X-ray plain film and MRI can diagnose the typing of CARM accurately, but MRI is also able to diagnose the fistula, visceral, spinal cord lesion and other abnormalities accompanied with CARM, while reducing the dose of X-ray radiation and damage in children, thus has higher clinical application value compared with inverted X-ray plain film.

Objective: To obtain polycystin-1 intracellular region. Methods: cDNA of polycystin-1 intracellular region was generated by PCR and then cloned into pProEX Hta, which was prokaryotic expression vector. After verified by sequencing, the recombinant was transformed into E.coli host to express and purify the fusion protein by affinity chromatography. Results: 660 bp cDNA of polycystin-1 intracellular region and 2.6×104 fusion protein were obtained. Conclusion: The fusion protein containing polycystin-1 intracellular region is obtained and is helpful for preparing anti-polycystin-1 monoclonal antibody.

Objective: To obtain polycystin-1 intracellular region. Methods: cDNA of polycystin-1 intracellular region was generated by PCR and then cloned into pProEX Hta, which was prokaryotic expression vector. After verified by sequencing, the recombinant was transformed into E.coli host to express and purify the fusion protein by affinity chromatography. Results: 660 bp cDNA of polycystin-1 intracellular region and 2.6×104 fusion protein were obtained. Conclusion: The fusion protein containing polycystin-1 intracellular region is obtained and is helpful for preparing anti-polycystin-1 monoclonal antibody.

Objective Rapamycin can improve characteristic pathology of AD by improving the level of autophagy.But, its internal mechanism still needs further study.This study was aimed to observe the protective effect of Rapamycin (RAPA) on the injury of rat pheochromocytoma (PC12) cells induced by β-amyloid protein25-35 (Aβ25-35).Methods PC12 cells in the logarithmic phase were randomly divided into 5 groups: control group(similar free-serum DMEM), model group, 10μmol/L RAPA treated group, 40μmol/L RAPA treated group and 160μmol/L RAPA treated group(add 10μmol/L, 40μmol/L RAPA, 160μmol/L respectively).Except the control group, each group was cultured with 20μmol/L Aβ25-35 to established the cell injury model.Results ①Compared with the survival rate of cells[(51.47±2.59)%] and the apoptosis rate of cells[(52.22±2.33)%] of the model group,the survival rate of cells in 10、40、160μmol/L RAPA treated groups and control group[(54.64±2.42)%, (64.79±2.91)% ,(56.50±2.55)% and (99.98±0.73)%] significantly increased, but the apoptosis rate of cells [(45.33±2.83)%, (36.89±2.85)%, (48.00±2.83)% and (3.33±2.45)%] significantly decreased(All P<0.05).②In model group,the expressions of p-PKB is 0.33±0.01, p-mTOR is 1.97±0.05, p-tau is 2.09±0.19.Compared with model group, in 10、40、160μmol/L RAPA treated groups and control group,these expressions of p-PKB (0.37±0.01, 0.42±0.01, 0.40±0.01 and 0.44±0.02) were significantly increased, however p-mTOR (1.64±0.05, 0.66±0.04, 0.35±0.01 and 0.62±0.01) and p-tau (2.02±0.15, 1.79±0.05, 1.86±0.06 and1.53±0.04) were decreased(All, P<0.05).ConclusionRAPA can increase Aβ25-35-induced PC12 cells viability, decrease cells apoptosis rates, and have a protective effect on Aβ25-35-induced PC12 cells death.The mechanism of its protective effect may be related to the inhibition of mTOR regulating PI3K/PKB/mTOR signal transduction pathway by negative feedback and the reduction of tau protein hyperphosphorylation.

Objective To study the protective effect of chlorogenic acid(CGA)on the apoptosis of PC12 cells induced byβ-amyloid protein23-35(Aβ25-35)and its mechanism. Methods The cells model of death was estab-lished by Aβ25-35 (20 μmol/L)-induced PC12 cells. The cells were interfered with 5 different concentrations of CGA. CCK-8 assay was used to detect cells viability to determine the 3 concentrations of CGA in future experi-ments. The cells were divided randomly into control group ,model group and interference groups with 3 different concentrations of CGA. Cells apoptosis rates were detected by flow cytometry;colorimetry method was used to detect MDA,SOD and GSH-Px. The mitochondrial membrane potential(MMP)was detected by fluorescent staining and the expression of caspase-3 by western blot. Results Compared with model group,the cells viability of CGA groups were increased but the apoptosis rates were reduced;the activity of SOD and GSH-Px were increased but the level of MDA,MMP and caspase-3 were decreased(P<0.05). Conclusions CGA has a protective effect on Aβ25-35-induced PC12 cells apoptosis and it may be related to the improvement of cellular antioxidation capacity and mitochondrial damage.