AIM:To observe the effects of granulocyte colony-stimulating factor (G-CSF) on calcium, sodium and potassium ion channel currents of the ischemic atrial myocytes in guinea pig by whole-cell patch clamp technique.METHODS:The guinea pig atrial myocytes were obtained by enzymolysis.Under ischemia and hypoxia condition, whole-cell patch clamp was used to observe the effects of G-CSF at various concentrations on the changes of the I-V curve, activation curve and availability of L-type calcium channel current (ICa,L) and voltage-dependent sodium channel current (INa), as well as I-V curve of delayed rectifier potassium channel current (IK).RESULTS:Under ischemic condition, the I-V curves of ICa,L were changed by acute G-CSF intervention in a dose-dependent fashion.Except for G-CSF at dose of 300 μg/kg, the other concentrations of G-CSF did not change the activation curve and availability of ICa,L, indicating that the effects of G-CSF on ICa,L were in a voltage-independent fashion.The I-V curves of ICa,L under ischemic condition were gradually approaching the normal levels by the higher dose of G-CSF, while the effect of 300 μg/kg G-CSF on ICa,L was similar to 100 μg/kg G-CSF.Acute G-CSF intervention at different doses did not change I-V curve, activation curve, and availability or steady-state availability of INa.As a part of IK, the rapid activating component (IKr) was improved by 100 μg/kg and 300 μg/kg G-CSF intervention with the similar effects, while the slowly activating component (IKs) was not changed by G-CSF.CONCLUSION:G-CSF affects ion channel electrophysiological properties of ischemic atrial myocytes in a voltage-independent but concentration-dependent manner, thus reducing the incidence of atrial arrhythmia.

OBJECTIVETo study the molecular mechanism of apoptosis of leukemic cells (K562 cells) induced by iron chelating agent deferoxamine (DFO).

METHODSThe exponentially growing K562 cells were used (1×10(6)/mL) in this study. The K562 cells were treated with different concentrations of DFO (10, 50 and 100 mmol/L), DFO+FeCl3 (10 μmol/L each) or normal saline (blank control). The cellular labile iron pool was measured with a fluorimetric assay using the metalsensitive probe calcein-AM. The viable count and cell viability were determined by typanblue assay. Cell apoptosis was determined by morphological study and flow cytometry assay. Caspase-3 activity in K562 cells was detected by colorimetry.

RESULTSAfter DFO treatment, the cellular labile iron pool and the viability of K562 cells were reduced and the cell apoptosis increased in a time- and dose-dependent manner compared with the blank control group. The apoptosis rate of K562 cells in the DFO+FeCl3 treatment group was not significantly different from that in the blank control group. The caspase-3 activity in K562 cells increased significantly 24 hrs after 50 and 100 μmmol DFO treatment when compared with the blank control group (P<0.01). There was a negative correlation between cellular labile iron pool and caspase-3 activity of K562 cells (r=-0.894, P<0.05).

CONCLUSIONSDFO induces apoptosis of leukemic cells possibly through decreasing cellular labile iron pool and increasing caspase-3 activity of the cells.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; Flow Cytometry ; Humans ; Iron Chelating Agents ; pharmacology ; K562 Cells

Objective To observe the relationship between apoptosis of K562 cell induced by iron-deprivation and activation of Caspase-3.Methods K562 cells were treated with desferrioxamine(DFO) in different dosages were collected at different time points.K562 cells were labelled with Annexin V/PI,and then the rate of apoptosis was measured by flow cytometry;The activation of Caspase-3 were detected by colorimetric method with pAN labelled substrate;The active protein of Caspase-3 were analyzed by Western blot.Results When K562 cells were treated with different concentrations of DFO,the apoptosis rate and the activity of Caspase-3 increases gradually.When K562 cells were incubated with DFO(50 ?mol/L and 100 ?mol/L) 24 h later,the enzymatic activity of Caspase-3 increases dramatically more than that of control group,and the difference was significantly(P0.05).All those effect above can be counteracted by equal mole concentration of FeCl_3.Conclusion Iron-deprivation maybe induce the apoptosis of K562 cell by chelating intracellular iron and activing Caspase-3.

To explore a rapid and easy method to detect labile iron of pool (LIP) in cells, HL-60 and K562 cells were cultured at a concentration 1 x 10(6)/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The iron deprivation was induced by adding desferrioxamine (DFO) 10 - 100 micromol/L for 0 - 48 hours. The intracellular LIP was measured by probe calcein-AM. Calcein fluorescence was monitored in 1420 multilabel counter. The results indicated that when HL-60 and K562 cells were incubated with different concentrations of DFO, the calcein fluorescence intensity was higher than that of control group at 12, 24 and 48 hours (P < 0.05). Fluorescence value of representing LIP in DFO groups was lower than that in the control group. In conclusion, DFO can decrease LIP in leukemia cells. The approach used in this study may provide a simple and reliable method for detection of intracellular iron homeostasis.
Cation Transport Proteins ; antagonists & inhibitors ; biosynthesis ; metabolism ; Deferoxamine ; pharmacology ; Fluoresceins ; Fluorescent Dyes ; HL-60 Cells ; Humans ; Iron ; metabolism ; Iron Chelating Agents ; analysis ; metabolism ; Iron-Regulatory Proteins ; metabolism ; K562 Cells

OBJECTIVETo analysis the homology among the extended-V region of the surface proteins in different serotype Streptococcus mutans (c, f, d, g) and to find out it's significance in anti-caries vaccine.

METHODSThe DNA of the bacteria (standarded serotype c, d, f, g and partial serotype c clinicals) was extracted and the extended-V region (SrV+, 1 384-2 514 bp) was amplified using polymerase chain reaction (PCR). Then the products were assessed using restriction fragment length polymorphism (RFLP) by endonuclease Dde I. The genotypings were sequenced and analysised using the program of BLAST on NCBI Gene Bank database.

RESULTSAbout 1.13 kb fragments were produced both in serotype c and f, the serotype d and g were failed. The RFLP results showed that five different patterns(A, B, C, D, E) among the 117 PCR products were reveled by Dde I. The ration of the genotypings A and B were the most among the strains, the C was lower, the D and E respectively was 1 and 3 strains per genotype. OMZ175 (serotype f) was belong to B genotype. Selected one of the A, B, C genotypings to sequenced and blasted. Then the results of the blastn showed that the identities of the gene sequence were 92%-98% between the serotype c and serotype f, part sequence of the serotype g was homology with the SrV+ of the serotype c, the protein sequence among serotype c, d, f, g were 77%-82%.

CONCLUSIONIt is reasonable to use some putative pipetides to study the anti-caries vaccine among the extended-V regions of the surface proteins in different serotype (c, d, f, g) in S. mutans.

DNA, Bacterial ; Genotype ; Membrane Proteins ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Serogroup ; Streptococcus mutans

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

OBJECTIVETo analyze the related factors of neck recurrence and regularity of cervical lymph nodes metastasis of pathologically node positive (pN+) tongue squamous cell carcinoma(SCC) and explore the neck treatment strategy for pN+ tongue SCC.

METHODSClinical and follow-up data of 138 patients with pN+ oral tongue SCC from Jan. 1991 to Dec. 2008 were reviewed. Distribution of neck metastatic and recurrent lymph nodes were analyzed. The influencing factors of neck recurrence of pN+ tongue SCC were analyzed.

RESULTSAll patients were followed over two years or until death. Using Kaplan-Meier method, the 3-year and 5-year overall survival rates were 46.4% and 36.2% respectively. Two hundred and three levels of 138 patients had metastasis and the involvement frequency of ipsilateral I, II, III reached to 94.6%. Sixty-six levels of 47 patients had neck recurrences and the involvement frequency of ipsilateral I, II, III reached to 77.3%. pT stage, pN stage, pTNM stage, extracapsular spread (ECS) of cervical lymph nodes were relevant to the neck recurrence of pN+ tongue SCC (all P < 0.05). When ECS of cervical lymph nodes was present, the neck recurrence rate of patients with postoperative radiation was lower than patients without postoperative radiation, but P value failed to demonstrate significant difference (P = 0.076). There were no significant difference of neck recurrence rates between different neck dissection methods (P > 0.05). Multivariate Cox analysis showed that pTNM stage and ECS of cervical lymph nodes were the independent prognostic factors of pN+ oral tongue SCC.

CONCLUSIONSpT stage, pN stage, pTNM stage, ECS of cervical lymph nodes were the influencing factors of neck recurrence of pN+ tongue SCC. Postoperative radiation may reduce the neck recurrence rate when ECS was present. There was no difference of the neck recurrence rate between modified neck dissection (MRND) and radical neck dissection (RND) and when the non-lymphatic structures were not involved, MRND should attempted. Metastatic and recurrent lymph nodes of pN+ tongue SCC were mostly distributed in ipsilateral I, II, III level and selective neck dissection (SND) can be applied to pN+ tongue SCC.

Carcinoma, Squamous Cell ; Humans ; Lymphatic Metastasis ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Retrospective Studies

Adult ; Catheter Ablation ; methods ; Female ; Humans ; Intervertebral Disc Displacement ; surgery ; Low Back Pain ; surgery ; Male ; Middle Aged

The clinical evaluation methods of facial paralysis can be divided into functional evaluation scales,neuro-electrophysiologi-cal tests and computer evaluation systems.The commonly used function evaluation scales include House-Brackmann Grading Scale(HB-GS),Burres-Fisch Facial Nerve Scoring System,Nottingham System,Sunnybrook facial grading System(SFGS),Degree of Facial Nerve Paralysis Hierarchical Scale,Facial Disability Index(FDI)and Facial Clinimetric Evaluation(FaCE)Scale,etc.Neuro-electrophysiological tests mainly consist of facial electromyography (EMG), electroneurography (ENoG), blink reflex (BR), and neural excitatory test (NET), etc.The computer evaluation system based on the sensor is mainly divided into the computer evaluation system based on infrared thermal image technology and the computer evaluation system based on biomedicine image recognition.This article briefly summarized the existing methods of facial paralysis evaluation in terms of sensitivity,stability,accuracy,ease of operation and economics.

Objective To investigate the accuracy and comparability of ?-glutamyltransferase (?- GT) measurement results on human serum samples and controI materials before and after calibration with a common human serum calibrator.Methods A human serum calibrator was prepared by pooling fresh serum aliquots and assigning a value for ?-GT with the IFCC reference method.The calibrator together with 5 human serum samples and 10 control samples were sent to 15 clinical laboratories and the sermn and control samples were measured with different analytical systems before and after a calibration with the calibrator.The results were analyzed for biases and interlaboratory variations.Results For the serum samples,the calibration resulted in reductions in biases from -9.0%～-14.2% to -0.8%～-7.9%,and in interlaboratory variations from 6.9%～11.6% to 2.8%～4.4%.No improvement was observed on the control samples.Conclusions Accuracy and comparability of serum ?-GT measurements can be improved by using a common human serum calibrator.Some control materials may not be commutable for human serum in ?-GT measurements.