The purpose of this study was to investigate the expression feature of a human tumor related gene chp2 in leukemia primary cells and leukemia cell lines, real-time quantitative PCR (RQ-PCR) was performed to detect the expression level of chp2 gene in peripheral blood mononuclear cells from 10 healthy individuals (as control) and 24 cases of leukemia, and in 4 kinds of leukemia cell lines. The results showed that the detection rate of chp2 gene in 10 normal controls was 80%, positive expression was (0.744 +/- 0.682) x 10(5) cps/microl. The expression levels of chp2 mRNA leukemia primary cells and leukemia cell lines were significantly higher than that in the normal control (p < 0.05). The expression levels of chp2 mRNA were higher in AML cells (7 cases), CML cells (6 cases), ALL cells (7 cases) and CLL cells (4 cases), and their expression levels were (11.637 +/- 5.588), (6.122 +/- 3.785), (4.262 +/- 2.561) and (3.434 +/- 1.974) x 10(5) cps/microl respectively. Gene chp2 positively expressed in four kinds of leukemia cell lines, and the expression levels in K562 cells, Jurkat cells, HL-60 cells and M07e cells were (5.243 +/- 1.852), (4.463 +/- 1.621), (4.137 +/- 1.837) and (2.578 +/- 1.137) x 10(6) cps/microl respectively. The expression level in leukemia cell lines was higher than that in primary cells. It is concluded that the human tumor related gene chp2 expression in leukemia primary cells and leukemia cell lines significantly increase, which may play an important role in growth process of leukemia cells.
Calcium-Binding Proteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Jurkat Cells ; K562 Cells ; Leukemia ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.
Bioreactors ; microbiology ; Colony Count, Microbial ; Escherichia coli ; genetics ; metabolism ; Hepatitis E virus ; genetics ; Nucleocapsid Proteins ; biosynthesis ; genetics ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Benzamides ; pharmacology ; Cation Transport Proteins ; antagonists & inhibitors ; metabolism ; Drug Resistance, Neoplasm ; drug effects ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; Imatinib Mesylate ; Imidazoles ; pharmacology ; K562 Cells ; MAP Kinase Signaling System ; Piperazines ; pharmacology ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; metabolism ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo assess agreement between the ultrasonic cardiac output monitor (USCOM) and conventional echocardiography (ECHO) in the measurement of cardiac output in newborn infants, investigate the accuracy and clinical utility of the USCOM in healthy neonates. To explore a more convenient, faster, more accurate hemodynamic monitoring method, for improving the outcome of the critically ill neonates.

METHODFrom October 1(st), 2011 to March 31(st), 2012, a total of 49 infants were included, 20 were term infants, 29 were preterm infants. Cardiac outputs were measured by both ultrasonic cardiac output monitor and echocardiography in all the infants, 60 times measurements were done in both the term infants the preterm infants. The cardiac output of the left and right ventricles, heart rate, diameter and velocity time integral of the aortic valve and pulmonary artery valve of each infant were recorded. The consistency of two methods was analyzed as described by Bland-Altman.

RESULTTerm the term infant group includea 20 term infants, 11 were male and 9 were female, the mean gestational age were (38.1 ± 0.56) weeks, mean age were (2 ± 1) days, mean weight were (3.2 ± 0.29) kg, mean Apgar score were 10. The mean left ventricular output measured by Echo was (242.3 ± 38.9) ml/(kg·min), measured by USCOM was (211.7 ± 38.5) ml/(kg·min); The mean right ventricular output measured by ECHO was (318.9 ± 47.0) ml/(kg·min), measured by USCOM was (340.7 ± 76) ml/(kg·min). Agreement between Echo and USCOM for left ventricular output (LVO) was (bias, ± limits of agreement, mean % error): (30.6 ± 51.1) ml/(kg·min), 21%, and for right ventricular output (RVO): (-21.8 ± 105) ml/(kg·min), 33.2%. The diameter of the aortic valve and pulmonary artery valve measured by conventional echocardiography were significantly larger than that estimated by ultrasonic cardiac output monitor (P < 0.001). The velocity time integral of the pulmonary artery valve measured by ultrasonic cardiac output monitor were significantly larger than measured by conventional echocardiography (P < 0.001). The heart rate, velocity time integral of the aortic valve measured by two methods had no significant differences (P > 0.05). The preterm neonates group included 29 preterm infants, 18 were male and 11 were female, the mean gestational age were (32.6 ± 2.8) weeks, mean age were (2 ± 1) days, mean weight were (1.88 ± 0.57) kg. All the infants were diagnosis as preterm infant, low birth weight. The mean left ventricular output measured by ECHO was (259.8 ± 70) ml/(kg·min), measured by USCOM was (235.6 ± 61.8) ml/(kg·min), the mean right ventricular output measured by ECHO was (318.9 ± 47.0) ml/(kg·min), measured by USCOM was (340.7 ± 76) ml/(kg·min). Agreement between Echo and USCOM for left ventricular output (LVO) was (bias, ± limits of agreement, mean % error): (24.1 ± 71.2) ml/(kg·min), 27.4%, and for right ventricular output (RVO): (-29.5 ± 192.9) ml/(kg·min), 51.8%. The diameter of the aortic valve and pulmonary artery valve measured by conventional echocardiography were significantly larger than estimated by ultrasonic cardiac output monitor (P < 0.001). The velocity time integral of the pulmonary artery valve measured by USCOM were significantly larger than that measured by conventional echocardiography (P < 0.001). The heart rate, velocity time integral of the aortic valve measured by two methods had no significant differences (P > 0.05).

CONCLUSIONAgreement between USCOM and conventional ECHO in the LVO measurement is acceptable, both in the term group and the preterm group. LVO measurement measured by USCOM is recommended. The accuracy and clinical utility of the USCOM in neonates is acceptable. USCOM is a convenient, fast and accurate hemodynamic monitoring method in neonates. While the agreement between USCOM and conventional ECHO in the RVO measurement is poor, especially in the preterm group, the results of the RVO cannot be considered interchangeable in the two methods.

Cardiac Output ; Echocardiography, Doppler ; instrumentation ; methods ; Female ; Heart Rate ; physiology ; Hemodynamics ; physiology ; Humans ; Infant ; Infant, Newborn ; Infant, Premature ; Intensive Care, Neonatal ; Male ; Monitoring, Physiologic ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Ventricular Function ; physiology

Abnormalities, Multiple ; diagnosis ; surgery ; Anus, Imperforate ; surgery ; Colon ; abnormalities ; Female ; Follow-Up Studies ; Humans ; Infant, Newborn ; Ischium ; abnormalities ; Treatment Outcome ; Twins, Conjoined ; surgery

The objective of this study was to compare the effects between knocking-out Sam68 gene by homologous recombination method and silencing the gene by siRNA silencing technique in DT40 cell line. Gene targeting technique was used to isolate Sam68 gene-deleted chicken DT40 cells. Meanwhile, Sam68 gene silencing cells was obtained by using stable expression of siRNA plasmid pSilencer-Sam68. Then, the function of these two cell lines were analyzed by comparing with wild-type DT40 cell line. The results showed that the growth retardation in Sam68 gene knocked-out cell line was observed due to elongation of the G2/M phase, but which could not be found in Sam68 gene silencing cell line. It is concluded that in accordance with study of protein function in living cells, use of gene knockout technique for cell line can provide the experimental results more real than those resulting from gene silence technique.
Animals ; Cell Line, Tumor ; Chickens ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Gene Silencing ; Gene Targeting ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo evaluate the efficacy of the monotherapy of 15 agents in treating essential hypertension.

METHODSAfter 2-week wash-out, a total of 370 patients with seated diastolic blood pressure 95-114 mmHg and seated systolic blood pressure < 180 mmHg were randomized to different therapeutic groups. 24-hour ambulatory blood pressure monitoring was performed before medication and at the end of 8 weeks.

RESULTAll the agents significantly reduced the 24 hour mean blood pressures after treatment except doxazosin, terazosin, and torasemide.

CONCLUSIONThe result suggested that the angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, beta-blockers and long-acting calcium antagonists were effective in treating essential hypertension, while the low-dose doxazosin, terazosin and torasemide can be used for combination therapy but not for monotherapy.

Adrenergic beta-Antagonists ; therapeutic use ; Angiotensin Receptor Antagonists ; Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Antihypertensive Agents ; classification ; therapeutic use ; Blood Pressure Monitoring, Ambulatory ; Calcium Channel Blockers ; therapeutic use ; Doxazosin ; therapeutic use ; Drug Therapy, Combination ; Humans ; Hypertension ; drug therapy ; Prazosin ; analogs & derivatives ; therapeutic use ; Sulfonamides ; therapeutic use ; Treatment Outcome

The aim of this study was to investigate the effect of intracellular acidification on the P-gp in K562/A02 cells. Confocal laser microscope was used to determine the intracellular acidification. MTT assay was used to detect the cytotoxicity of intracellular acidification on K562 and K562/A02 cells. Flow cytometry was applied to measure the influence of intracellular acidification on the activity of P-gp. The P-gp expression at protein and mRNA levels were determined by Western blot and real-time RT-PCR respectively. The results indicated that intracellular acidification had no obvious cytotoxicity on K562 and K562/A02 cells. The function of P-gp in K562/A02 cells weakened along with decrease of intracellular acidification, the intracellular acidification significantly increased the accumulation of Rhodamine 123 (Rh 123) and suppressed the efflux of Rh 123 mediated by P-gp. The intracellular acidification also inhibited the expression of P-gp in K562/A02 cells at protein and mRNA levels which showed intracellular acidification with time-dependence. It is concluded that the intracellular acidification can inhibit the expression and function of P-gp in K562/A02 cells.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; Hydrogen-Ion Concentration ; K562 Cells