This study was aimed to investigate the effects of bortezomib combined with 5-azacytidine on the apoptosis of K562 cells and expressiom of SHIP mRNA. The K562 cells were cultured and treated with different concentrations of bortezomib, 5-azacytidine or their combination for 24 hours. Then, the expression of SHIP mRNA was detected by RT-PCR,the cell proliferation was analyzed by using MTT assay and flow cytometry. The results showed that 5-20 nmol/L bortezomib could effectively inhibit the proliferation of K562 and this inhibitory effect gradually enhanced along with the increase of bortezomib concentration, the group of bortezomib combined with 5-azacytidine showed more inhibitory effect on K562 cells than that of bortezomib or 5-azacytidine alone.The bortezomib could promote the apoptosis of K562 cells in a dose-dependent manner,and this apoptotic effect was higher in group of bortezomib combined with 5-azacytidine than that in group of bortezomib or 5-azacytidine alone.Bortezomib could down-regulated the expression of SHIP mRNA in a dose-dependent manner,and this down-requlated effect was higher in group of bortezomib combined with 5-azacytidine than that in group of bortezomib or 5-azacytidine alone.It is concluded that bortezomib and 5-azacytidine can induce apoptosis by inhibiting the expression of SHIP mRNA in K562 cells.The combination of bortezomib with 5-azacytidine displays a synergetic effect.
Apoptosis ; drug effects ; Azacitidine ; pharmacology ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Proliferation ; Humans ; Inositol Polyphosphate 5-Phosphatases ; K562 Cells ; Phosphoric Monoester Hydrolases ; genetics ; Pyrazines ; pharmacology ; RNA, Messenger ; biosynthesis

OBJECTIVETo detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.

METHODSUsing the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).

RESULTSAmong the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.

CONCLUSIONNot only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; genetics ; Humans ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phosphoric Acids ; Polymerase Chain Reaction ; Rifampin ; pharmacology

OBJECTIVETo compare the three dimensional morphology and surface roughness changes of enamel eroded for different etching time.

METHODSFifteen freshly extracted sound human pre-molars for orthodontic purpose were collected. The buccal surface of teeth were prepared into smooth enamel slices, and then were randomly divided into 5 groups based on their etching time 0 s (control group), 5 s, 10 s, 20 s, 30 s, respectively by 37% phosphoric acid. The three dimensional morphology was observed under atomic force microscope (AFM). The profile was analyzed, and the value of Ra, Rq, Rz and the surface area and volume were measured.

RESULTSThe AFM photograph showed that with the etching time from 0 s to 20 s the enamel surface demineralised gradually, the top structure of enamel rod and the fish scaled structure became obvious. But the morphology only changed a bit after 20 s. The surrounding inter-rod enamel eroded first, the depth increased to 2.8 µm at 20 s but decreased to 1.8 µm at 30 s. The value of Ra increased from (19.69 ± 3.42) nm to (359.51 ± 75.79) nm, and Rq from (22.02 ± 5.57) nm to (431.02 ± 83.09) nm, Rz from (0.24 ± 0.08) µm to (2.38 ± 0.26) µm. Except for groups 20 s and 30 s, the difference among other groups was statistically significant (P < 0.05). The surface area expanded from (406.77 ± 3.88) µm(2) to (546.69 ± 84.02) µm(2), and surface volume from (65.73 ± 14.46) µm(3) to (474.63 ± 52.50) µm(3).

CONCLUSIONSThe depth, surface roughness, surface area and volume caused by erosion increased with etching time. The three dimensional morphology greatly changed by acid-etching process.

Acid Etching, Dental ; methods ; Analysis of Variance ; Dental Enamel ; drug effects ; ultrastructure ; Humans ; Microscopy, Atomic Force ; Phosphoric Acids ; pharmacology ; Random Allocation ; Surface Properties ; drug effects ; Time Factors

This study was aimed to investigate the effects of proteasome inhibitor bortezomib on proliferation, apoptosis and the SHIP expression of K562 cells. K562 cells were treated with bortezomib of different concentrations. Cell proliferation was analyzed by MTT assay, cell apoptosis was detected by flow cytometry and SHIP mRNA expression was assayed by RT-PCR.The results showed that after being treated with 10, 20, 50 and 100 nmol/L bortezomib for 24 h, the inhibitory rates of K562 cells were (5.76 ± 1.47)%, (10.55 ± 1.59)%, (17.14 ± 2.05)% and (27.69 ± 3.57)% respectively, and were higher than that in control (1.30 ± 0.10); when K562 cells were treated with 20 nmol/L bortezomib for 24, 48 and 72 h, the inhibitory rates of cell proliferation were (10.55 ± 1.59)%, (16.33 ± 2.53)% and (19.78 ± 1.56)% respectively, there was statistic difference of cell proliferation rate between 24 h group and 48 h group (P < 0.05). After being treated with 10,20,50,100 nmol/L bortezomib for 24 h, the apoptotic rates of K562 cells were (12.7 ± 0.6)%, (26.9 ± 0.9)%, (32.6 ± 1.2)% and (72.5 ± 1.5)% respectively,and all higher than that in control (1.0 ± 0.5)% (P < 0.05). According to results of RT-PCR detection, the expression level of SHIP mRNA was obviously up-regulated after treatment with bortezomib, and showed statistical difference in comparison with control. It is concluded that bortezomib inhibits proliferation of K562 cells in time and concentration-dependent manner and induces apoptosis through up-regulation of SHIP gene.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Proliferation ; drug effects ; Humans ; Inositol Polyphosphate 5-Phosphatases ; K562 Cells ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Proteasome Inhibitors ; pharmacology ; Pyrazines ; pharmacology

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.
Binding, Competitive ; Catalytic Domain ; Crystallography, X-Ray ; Dose-Response Relationship, Drug ; Drug Evaluation, Preclinical ; Edetic Acid/pharmacology ; Enzyme Inhibitors/*pharmacology ; Human ; Inhibitory Concentration 50 ; Kinetics ; Models, Molecular ; Molybdenum/*pharmacology ; Phosphoric Acids/*pharmacology ; Protein Structure, Tertiary ; Protein-Tyrosine-Phosphatase/*antagonists & inhibitors/*chemistry/isolation & purification ; Substrate Specificity ; Tungsten Compounds/*pharmacology