This study was purposed to investigate the differences of cyto biological characteristics and protein expression levels between bortezomib-resistant T-lymphoblastic lymphoma/leukemia cell lines JurkatB containing PSMB5 G322A mutation and their parent cell line Jurkat, The cytotoxicities of bortezomib and chemotherapeutic drugs to JurkatB5 cells (end selection concentration of bortezomib was 500 nmol/L), JurkatB8 (end selection concentration 800 nmol/L) and Jurkat cells were analyzed. The cell growth curves were drawn with viable cell counts by trypan blue assay, the colony formation rate were assayed by soft-agar colony culture, and the cell distributions in cell cycle were analyzed by flow cytometry, mRNA expression levels of multidrug resistance (MDR) genes MDR1, LRP and MRP were measured by real-time fluorescence quantitative RT-PCR, the differences of protein expression levels were detected by SpringBio antibody microarray containing 720 proteins. The results showed that the drug resistance multiples for 48 hours of JurkatB5 and JurkatB8 cells (relative to Jurkat) to bortezomib were increased by 33.52 and 39.04 times, respectively. JurkatB5 and JurkatB8 cells did not display significant cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide after exposure for 48 hours. There were no significant differences in the cell growth curve, colony formation rate and cell distributions in cell cycle between JurkatB5, JurkatB8 and Jurkat cells (p > 0.05). There were no significant differences of mRNA expression levels of MDR1, LRP, MRP between JurkatB5 and Jurkat cells (p > 0.05). There were 264 analyzable expression points detected by antibody microarray. Among them, 252 protein expression levels were not significantly different between JurkatB5, JurkatB8 and Jurkat cells (< 2-fold), including 15 drug resistance-related proteins. 12 proteins were detected at higher or lower expression levels in JurkatB5 or JurkatB8 cells then that in Jurkat cells (cell division cycle protein 34, cell division cycle protein 37, CD34 Type II, matrix metalloproteinase-2, tenascin, Golgi complex, involucrin, histone deacetylase 1, perforin, prolactin, retinoic acid receptor β, integrin β-1), but no proteins were detected in JurkatB5 and JurkatB8 cells with higher or lower expression levels than that in Jurkat cells. It is concluded that there are no significant differences in the characteristics of cellular biology between Jurkat and JurkatB with bortezomib-resistant and PSMB5 G322A mutation. There are no significant phenotype change of MDR and overexpression of genes related to MDR in PSMB5 mutated cells. There are no significantly differential expressions of a majority of known proteins related to drug resistance, tumor cells growth, proliferation, apoptosis, malignancy degree, aggressiveness.
Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Drug Resistance, Multiple ; genetics ; Gene Expression Profiling ; Humans ; Jurkat Cells ; Mutation ; Proteasome Endopeptidase Complex ; genetics ; Pyrazines ; pharmacology

OBJECTIVE Lychee seed, a famous traditional Chinese medicine, recently were reported to improve the learning and memory abilities in mice. However, it is still unclear whether lychee seed saponins (LSS) can improve the cognitive function and associated mechanisms. METHODS In present studies, we established the Alzheimer disease (AD) model by injecting Aβ25-35 into the lateral ventricle of rats. Then the spatial learning and memory abilities of LSS- treated rats were evaluated with the Morris water maze, meanwhile the protein expressions of AKT, GSK3β and Tau in the hippo?campal neuron were analyzed by immunohistochemistry and Western blotting. RESULTS The results showed LSS can improve the cognitive functions of AD rats through shortening the escape latency, increasing the number across the platform, platform quadrant dwell time and the percentage of the total distance run platform quadrant. The protein expression of AKT was significantly up-regulated and that of GSK3β and Tau were decreased remarkably in the hippocampal CA1 area. CONCLUSION Our study is the first to show that LSS significantly improve the cognitive function and prevent hippocampal neuronal injury of the rats with AD by activation of the PI3K/AKT/GSK3β signaling pathway, suggesting LSS may be developed into the nutrient supplement for the treatment of AD.

OBJECTIVETo develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU).

METHODSA recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six β-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for β-lactams was established with PBP2x* and HRP-conjugate.

RESULTSPBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment.

CONCLUSIONThis assay developed can detect all 16 β-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Animals ; Milk ; chemistry ; Penicillin-Binding Proteins ; metabolism ; beta-Lactams ; analysis ; metabolism

This study was aimed to investigate the reversible effect of tetrandrine, toremifene and their combination on multidrug resistance of K562/A02 cell line. The IC(50) (the concentration causing 50% inhibition of cell growth) of adriamycin (ADR) were assayed by MTT method, the expression of MDR1 mRNA was measured by RT-PCR, the concentration of p-glycoprotein (P-gp) and intracellular ADR were detected by flow cytometry. The results showed that the IC(50) of ADR on K562/A02 and K562 cells were 57.43 and 1.16 mg/L, respectively. The IC(50) of ADR on K562/A02 cells after treatment with tetrandrine, toremifene and both combination were 14.12, 20.74 and 9.14 mg/L respectively, but both drugs did not influence the IC(50) of ADR on K562 cells. Pretreating K562/A02 cells with toremifene (2.5 micromol/L), tetrandrine (1 micromol/L) or both for 72 hours partially restored the sensitivity of K562/A02 cells to ADR. Tetrandrine and toremifene (alone or combination) elevated the ADR concentration in K562/A02, down regulated the expressions of P-gp and MDR1 mRNA. It is concluded that multidrug resistance of K562/A02 cells can be partially reversed by tetrandrine or toremifene, the combination of both drugs shows a higher synergistic reversal effect.
Antineoplastic Agents, Hormonal ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Benzylisoquinolines ; pharmacology ; Doxorubicin ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Humans ; K562 Cells ; Toremifene ; pharmacology

Iron is an essential element for cell growing including tumor cells. This study was purposed to explore the effect of desferrioxamine (DFO) on cell line K562/A02 and its mechanism. K562/A02 cells were cultured with different concentrations of DFO. The inhibitory effects of adriamycin (ADM) used alone or combined with DFO on the proliferation of K562/A02 was evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treatment with 0, 12.5, 25 and 50 µmol/L DFO alone or in combination with 1 mg/L ADM were analyzed by flow cytometry. ADM accumulation in K562/A02 cells after treatment with different concentrations of 0, 12.5, 25 and 50 µmol/L DFO were also analyzed by flow cytometry. The levels of BAX/BCL-2 and MDR1 mRNA were determined by RT-PCR, and then the protein level of P-glycoprotein (P-gp) was detected by Western blot. The results showed that the IC(50) of ADM for K562 and K562/A02 cells were (1.46 ± 0.07) mg/L and (40.98 ± 3.05) mg/L respectively. The resistance of K562/A02 cells to ADM was 28.06 times as that of K562 cells. After treatment of K562/A02 cell with DFO of 12.5, 25 and 50 µmol/L for 48 hours, the resistance of K562/A02 cells to ADM were increased by 24.95, 16.11 and 9.99 times respectively. When K562/A02 cells were incubated with different concentrations of DFO of 12.5, 25, 50 µmol/L for 48 hours, the apoptosis rat were (3.50 ± 0.30)%, (7.27 ± 0.32)% and (12.53 ± 1.21)% respectively. After co-culture with DFO and ADM for 48 hours, apoptosis rate were (6.13 ± 0.29)%, (9.57 ± 0.40)% and (18.97 ± 1.10)% respectively. The above apoptosis rates was much higher than that of control group (p < 0.05) and they were dose-dependent. In comparison between DFO + ADM group and DFO group, there was no significant difference (p > 0.05). Expression rate of BAX/BCL-2 increased. The levels of MDR1 mRNA reduced. Furthermore, expression of P-gp also decreased in K562/A02 cells. It is concluded that iron increase can promote K562/A02 cells growth and inhibit their apoptosis. Otherwise, iron-deprivation can induce K562/A02 cells apoptosis. DFO disturbs the iron metabolism and inhibits DNA synthesis of K562/A02 cells. This action of DFO may enhance the susceptibility of K562/A02 cells to apoptosis induced by chemotherapeutic drugs. The iron-deprivation may play a role in the treatment of leukemia with combination of DFO with other anticancer agents.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; Deferoxamine ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Iron ; metabolism ; K562 Cells ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo search for an effective method of reducing intraoperative blood loss in radical retropubic prostatectomy (RRP).

METHODSWe performed RRP for 100 patients with prostate cancer, 50 (group A) with the Walsh or Poor method for handling the dorsal venous complex (DVC), and the other 50 (group B) through the following three additional procedures for hemostasis: first placing a #7 prophylactic suture in the distal position of DVC, then ligating the vascular bundle of the prostatic apex with continuous 4-0 Vicryl sutures, and lastly placing a 4-0 absorbable suture followed by freeing the neurovascular bundle (NVB) or freeing NVB before suturing the remained levator ani myofascia and the deep layer of Denovilliers' fascia above the rectal serosa with 4-0 Vicryl. We assessed the effects of the three hemostatic methods in RRP by comparing the volumes of intraoperative blood loss and transfusion, operation time and perioperative levels of hemoglobin.

RESULTSThere were no significant differences between groups A and B in age, PSA, Gleason score, clinical stage, prostate volume, operation time and perioperative hemoglobin levels (P>0.05). The volumes of intraoperative blood loss and transfusion were markedly higher in group A ([1103.00 +/- 528.03] ml and [482.00 +/- 364.60] ml) than in B ([528.00 +/- 258.96] ml and [140.00 +/- 266.28] ml) (P<0.05).

CONCLUSIONIntraoperative blood loss in RRP could be significantly decreased by placing a prophylactic hemostatic suture in the distal position of DVC, continuous suture of the vascular bundle of the prostatic apex after cutting off the urethra, and placing a fine absorbable suture above NVB or continuous suture of the remained levator ani mony fascia and the deep layer of Denovilliers'fascia above the rectal serosa with absorbable sutures after freeing NVB.

Aged ; Blood Loss, Surgical ; prevention & control ; Hemostatic Techniques ; Humans ; Male ; Middle Aged ; Prostatectomy ; methods ; Prostatic Neoplasms ; surgery

OBJECTIVETo investigate the role of platelet-derived growth factor-BB (PDGF-BB) in the degeneration of the hypertrophied ligamentum flavum (LF) in the lumbar spine.

METHODSSurgical specimens of degenerative hypertrophied LF were obtained from 11 patients with lumbar spinal stenosis (LSS, mean age 57.8 years), with those from 10 age-matched patients with lumbar disc herniation (LDH, mean age 53.5 years) as the normal controls. The thickness of the LF was measured preoperatively by axial T1-weighted magnetic resonance imaging (MRI) at the facet joint level. Immunohistochemistry was employed to determine the expression of PDGF β and PDGF-BB in the LF. The mRNA and protein expressions of PDGF-BB in the LF were detected using real-time PCR and Western blotting, respectively.

RESULTSThe thickness of the LF was 5.30±1.12 mm in the degenerative group and 2.80±1.53 mm in the control group, showing a significant difference between the two groups (P<0.001). PDGF-β and PDGF-BB were positive in the fibroblasts in hypertrophied LF. The mRNA and protein expressions of PDGF-BB were significantly higher in the degenerative group than in the control group (P=0.013 and 0.023, respectively).

CONCLUSIONThe high expression of PDGF-BB in the hypertrophied LF suggests its important role in the development of hypertrophy of LF in lumbar spinal canal stenosis.

Adult ; Aged ; Female ; Humans ; Hypertrophy ; metabolism ; Intervertebral Disc Degeneration ; metabolism ; pathology ; Ligamentum Flavum ; metabolism ; pathology ; Lumbar Vertebrae ; Male ; Middle Aged ; Proto-Oncogene Proteins c-sis ; metabolism ; Spinal Stenosis ; metabolism ; pathology

BACKGROUND5-dihydroxyanthraquinone-2-carboxylic acid (rhein) inhibits oxidoreduction induced by reducing nicotingamide adenine dinucleotide in the mitochondria and reducing reactive oxygen species, it also suppresses lipid peroxidation in rat brain homogenates. This study was to assess the effects of anthraquinone derivatives, rhein on synaptic transmission in the rat hippocampal CA1 pyramidal cell layer by intracellular recording.

METHODSThe excitatory postsynaptic potential (EPSP) evoked by stimulation of the Schaffer collaterals in the presence of bicuculline (15 micromol/L) was depressed by application of rhein (0.3 - 30 micromol/L). The amplitude of the EPSP was restored within 20 minutes after removal of rhein from the supernatant. At a concentration of 30 micromol/L, rhein reduced the amplitude of the EPSP to 42% +/- 3.7% (n = 24) of the control. Subsequently, wavelet spectral entropy was used to analyze the EPSP.

RESULTSA strong positive correlation was observed between the wavelet spectral entropy and other parameters such as amplitude, slope of rising phase and slope of descending phase of the EPSP. The paired-pulse facilitation (PPF) of the EPSP was significantly increased by rhein (30 micromol/L). The inhibitory postsynaptic potential (IPSP) recorded in the presence of CNQX (20 micromol/L) and APV (40 micromol/L) is not altered by rhein (30 micromol/L).

CONCLUSIONSRhein (30 micromol/L) can decrease the frequency but not the amplitude of the miniature EPSP (mEPSP). It is suggested that rhein inhibits excitatory synaptic transmission by decreasing the release of glutamate in rat hippocampal CA1 pyramidal neurons.

Animals ; Anthraquinones ; chemistry ; pharmacology ; Entropy ; Excitatory Postsynaptic Potentials ; drug effects ; Hippocampus ; drug effects ; physiology ; In Vitro Techniques ; Male ; Rats ; Rats, Wistar ; Synaptic Transmission ; drug effects

To study the effects of sodium valproate (VPA) on human myelodysplastic syndrome cell line MUTZ-1. The cell proliferation was determined by MTT assay, apoptotic morphological features were observed by light microscopy and transmission electronmicroscopy, cell apoptosis and cell cycle shift were analyzed by flow cytometry (FCM). The results showed that VPA could inhibit the growth of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in MUTZ-1 cells treated with 4 mmol/L VPA for 72 hours. Pyknosis of cells and nuclei, disintegration of nuclear chromatin and apoptotic body could be observed by light microscopy. Aggregation and margination of nuclear chromatin, concentration of plasm, increment of density and chromatin mass of irregular size could be observed by transmission electronmicroscope. The flow cytometric analysis indicated that the VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner, ratio of cells at G(0)/G(1) phase increased and ratio of cells at S phase decreased in dose-dependent manner, the cells were arrested at G(0)/G(1) phase. It is concluded that the VPA can induce apotosis and inhibite proliferation of MUTZ-1 cells via arresting cells at G(0)/G(1) phase.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Humans ; Myelodysplastic Syndromes ; pathology ; Valproic Acid ; pharmacology

The aim of this study was to analyze the hematopoietic chimerism after non-myeloablative allogeneic peripheral blood stem cell transplantation (NAPBSCT). 28 patients received NAPBSCT were evaluated. The conditioning regimen included FBC (fludarabine, busulphan, cyclophosphamide) +/- Ara-C. Peripheral blood was collected before and after transplantation in different periods. Semi-quantitative assessment of hematopoietic chimerism was performed by short tandem repeat-polymerase chain reaction (STR-PCR), polyacrylamide gel electrophoresis (PAGE) and silver staining, and analyzed by Image Analysis System. The results showed that on day 30 after transplantation, one patient failed to engraft, but 22 cases formed complete chimerism (CC) and 5 cases were of mixed chimerism. On day 7 after transplantation, the average percentage of donor cells was 74.71%. The time of dominance of the donor-specific allelic pattern preceded the recovery time of neutrophils and platelets. The incidence of aGVHD in group CC was significantly higher than that in group MC (P < 0.05). There was no significant difference in the incidence of cGVHD and disease relapse between group CC and group MC (P > 0.05). One patient relapsed in CC status without a transitional stage of MC. One patient with MC rejected grafts in early stage. 3 patients with MC transferred to CC and got complete remission after early implementation of therapy. It is concluded that sequential and quantitative detection of chimerism may be of great value to evaluate engraftment and to predict graft rejection, disease relapse and GVHD. Furthermore, it may provide a basis for early intervention treatment in the related complications.
Adolescent ; Adult ; Chimerism ; Female ; Graft vs Host Disease ; prevention & control ; Humans ; Leukemia, Myeloid, Acute ; therapy ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Transplantation Chimera ; blood ; genetics ; Transplantation Conditioning ; Transplantation, Homologous