mRNAs of alpha-adrenoceptor (α-AR) subtypes are found in neurons in dorsal root ganglion (DRG) and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve (CCI). Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.

OBJECTIVETo investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression.

METHODSThe small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay.

RESULTSThe transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively.

CONCLUSIONAt the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.

Apoptosis ; genetics ; Cell Proliferation ; Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; RNA, Small Interfering ; Transfection

OBJECTIVETo explore the modulatory effect of niflumic acid and blocker of calcium channel on the desensitization of gamma aminobutyric acid (GABA)-activated currents in dorsal root ganglion(DRG) neurons from rat.

METHODSThe whole-cell patch-clamp technique was used to observe the modulatory effect of niflumic acid and blocker of calcium channel on the desensitization of GABA-activated currents in neurons freshly dissociated from rat DRG neurons.

RESULTSApplication of GABA (0.1-1 000 micromol/L) could induce concentration-dependent inward currents in some cells (212/223, 95.11%). GABA-(100 micromol/L) activated currents was (1.32 +/- 0.74) nA (n = 84). However, pre-application of niflumic acid (1-100 micromol/L) and nitrendipine (specific blocker of L-calcium channel)(0.1-30 micromol/L) could inhibit the GABA-activated inward current which was identified to be GABAA receptor-mediated current. The inhibitory effects of niflumic acid and nitrendipine were concentration-dependent. The suppression rate of 10 micromol/L niflumic acid and nitrendipine to GABA-activated currents were (31.60% +/- 4.87%) (n = 19) and (43.60% < or = 5.10%) (n = 5), respectively. The desensitization of GABA-activated currents had double exponential characteristic. Tau value was (14.68 +/- 5.11) s (n = 6) and (175.8 +/- 42.67) s (n = 6, r = 0.9647), respectively. Pre-application of niflumic acid (100 micromol/L) and nickel chloride (nonspecific blocker of L-calcium channel) (100 micromol/L) altered tau value of the desensitization of GABA-activated currents, tau value reduced for (4.64 +/- 2.21) s (n = 3), (43.70 +/- 14.34) s ( n = 3, r = 0.9548) and (4.64 +/- 2.21) s (n = 3), (43.70 +/- 14.34) s (n = 3, r = 0.9721).

CONCLUSIONPre-application of niflumic acid exerts a more strong inhibitory effect on the peak value of GABA-activated current, which possibly is through blocking the calcium-activated chloride ion channel to accelerate the desensitization of GABA-activated currents.

Animals ; Animals, Newborn ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Ganglia, Spinal ; drug effects ; physiology ; Membrane Potentials ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Niflumic Acid ; pharmacology ; Nitrendipine ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; pharmacology

OBJECTIVETo explore the modulatory effect of niflumic acid (NFA) on gamma aminobutyric acid (GABA)-activated currents of dorsal root ganglion (DRG) neurons in rat.

METHODSThe whole-cell patch-clamp technique was used to record the NFA- and GABA-activated currents in neurons freshly dissociated from rat DRG neurons.

RESULTSApplication of NFA(0.1 - 100 micromol/L) could induce concentration-dependent outward currents in some cells (21/48,43.75%), and GABA (0.1 - 100 micromol/L) could induce concentration-dependent inward currents in some cells(150/159,94.32%). NFA-(100 micromol/L) and GABA-(100 micromol/L) activated currents were (0.27 +/- 0.06) nA (n = 12) and (1.29 +/- 0.72) nA (n = 53) respectively. However, pre-application of NFA (0.1 - 100 micromol/L) could inhibit the GABA-activated inward current which was identified to be GABAA receptor-mediated current. The inhibitory effects of NFA were concentration-dependent. NFA could not alter the EC50 (about 30 micromol/L) and inverse potential (about -10 mV) of GABA-activated current (P > 0.05).

CONCLUSIONPre-application of NFA exerts a more strong inhibitory effect on the peak value of GABA-activated current.

Animals ; Cell Separation ; Cells, Cultured ; Ganglia, Spinal ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Niflumic Acid ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; metabolism

mRNAs of alpha-adrenoceptor (α-AR) subtypes are found in neurons in dorsal root ganglion (DRG) and change after peripheral nerve injury. In this study, the distribution of α-AR subtype proteins was studied in L5 DRG of normal rats and rats with chronic constriction injury of sciatic nerve (CCI). Using immunofluorescence technique, it was found that α1A-, α1B-, and α2A-AR proteins were expressed in large, medium, and small size neurons in normal DRG, and significantly increased in all size neurons 14 days after CCI. α1D- and α2C-AR was also expressed in all size neurons in normal DRG. However, α1D-AR was significantly increased and α2C-AR was decreased in small size neurons 14 days post CCI. α2B-AR neurons were not detectable in normal and CCI DRG. Co-expression of α1A- and α2A-AR in the same neuron was observed in normal DRG and increased post CCI. Collectively, these results indicated that there is distinct distribution of α-AR subtypes in DRG neurons, and the distribution and levels of expression of α-AR subtypes change differently after CCI. The up-regulation of α-AR subtypes in DRG neurons may play an important role in the process of generating and transmitting neuropathic pain.
Animals ; Cell Size ; Chronic Disease ; Constriction, Pathologic ; Fluorescent Antibody Technique ; Ganglia, Spinal ; metabolism ; pathology ; Male ; Microscopy, Confocal ; Neurons ; metabolism ; pathology ; Pain Measurement ; methods ; Pain Threshold ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-1 ; metabolism ; Receptors, Adrenergic, alpha-2 ; metabolism ; Sciatic Nerve ; injuries ; surgery

Objective To study the pharmacodynamic material basis of licorice andimprove the role of sleep.Methods Thirty Wistar rats were divided into 3 groups, which were normal control group, licorice powder group, glycyrrhetinic acid group.The freeze-dried powder 20 g, water dissolved into 0.5 kg· L-1 solution, administered orally, the normal con-trol group were given equal volume of distilled water.One times a day, continuous 7 d.Blood transitional component were analzed by HPLC method.Results Glycyrrhetinic acid could make the peak area of endog-enous substances in cerebrospinal fluid ( CSF ) higher than the peak area of the blank, the peak area ratio of glycyrrhizin could make the endoge-nous substances in cerebrospinal fluid in the area is about 8.6 times peak area of the blank CSF.That the main chemical components of licorice glycyrrhetinic acid can significantly promote the secretion of endogenous substances in the cerebrospinal fluid, so as to improve the role of sleep.Conclusion By analysis and comparison, which indicates that the active constituent( glycyrrhetinic acid) in licorice are confirmed and could im-prove the sedative function.

OBJECTIVEBy inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle.

METHODSThe small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay.

RESULTSThe transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P < 0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively.

CONCLUSIONSThe synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.

Cell Cycle ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA Interference ; RUNX1 Translocation Partner 1 Protein ; Transfection

OBJECTIVETo establish and characterize a lung adenocarcinoma cell line from a female patient in Xuanwei, Yunnan province.

METHODSSurgical specimen of the lung adenocarcinoma was obtained and cultured immediately in RPMI 1640 medium with 10% fetal bovine serum and 10(5) U/L penicillin and 100 mg/L streptomycin. When stable proliferation of the cells was achieved after over 40 passages in culture, the biological features of the cell line were investigated by cell morphology, karyotyping, protein marker expression [cytokeratins (CKs), epithelial membrane antigen (EMA) and CD proteins], growth kinetics, cell cycle phase distribution, mitotic index, colony formation in soft agar, cell invasion and tumorigenicity in Balb/c nude mice.

RESULTSThe established cell line was stably cultured for over 80 passages during a one-year period as an anchorage-dependent monolayer of short spindle, polygonal to epithelioid cells under phase contrast microscope. Microglandular cavities and disordered microfilaments were observed under transmission electron microscope. The growth curve presented in an "S" shape with the cell population doubled every 46.7 hours. The mitotic index was 1.5% and the colony formation rate was 8.3%. The cell cycle distribution included 76.9% in G(0)/G(1), 15.1% in S and 8.0% in G(2)/M. The cell line displayed a hypotriploid karyotype with a mode of 66 chromosomes and a median of 64 chromosomes. The cells expressed CK7, CK8, CK (Pan) and EMA by immunohistochemistry. A high level of cell surface expression of CD13 and CD59 was evident by flow cytometry. The cells were able to penetrate Matrigel in vitro but failed to form a stable xenograft in nude mice.

CONCLUSIONA new human lung adenocarcinoma cell line, designated as XLA-07, is successfully established from a Xuanwei lung cancer patient.

Adenocarcinoma ; metabolism ; pathology ; Animals ; CD13 Antigens ; metabolism ; CD59 Antigens ; metabolism ; Cell Culture Techniques ; Cell Cycle ; Cell Line, Tumor ; ultrastructure ; Cell Proliferation ; Female ; Humans ; Karyotyping ; Keratins ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mucin-1 ; metabolism ; Neoplasm Transplantation ; Polyploidy ; Tumor Stem Cell Assay

Immobilized penicillin acylase was used for bioconversion of penicillin PG into 6-APA in aqueous two-phase systems consisting of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be about 5.78 in the presence of 1% NaCl. Enzyme kinetics showed that the reaction reached equilibrium at roughly 7 h. The 6-APA mole yields were 85.3% (pH 7.8, 20 degrees C), with about 20% increment as compared with the reaction of single aqueous phase buffer. The partition coefficient of PG (Na) varied scarcely, while that of the product, 6-APA and phenylacetic acid (PA) significantly varied due to Donnan effect of the phase systems and hydrophobicity of the products. The variation of the partition coefficients of the products also affected the bioconversion yield of the products. In the aqueous two-phase systems, the substrate, PG, the products of 6-APA and PA were biased in the top phase, while immobilized penicillin acylase at completely partitioned at the bottom. The substrate and PG entered the bottom phase, where it was catalyzed into 6-APA and PA and entered the top phase. Inhibition of the substrate and products was removed to result in improvement of the product yield, and the immobilized enzyme showed higher efficiency than the immobilized cells and occupied smaller volume. Compared with the free enzyme, immobilized enzyme had greater stability, longer life-time, and was completely partitioned in the bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtered 450 nm light, while pH-sensitive polymer PADB could be recovered at the isoelectric point (pH 4.1). The recovery of the two copolymers was between 95% and 99%.
Catalysis ; Enzymes, Immobilized ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Penicillanic Acid ; analogs & derivatives ; chemistry ; metabolism ; Penicillin Amidase ; metabolism ; Penicillin G ; chemistry ; metabolism ; Phase Transition ; Polymers ; chemistry ; Substrate Specificity

Aim To detect the expression of miRNA-99b and mTOR in glioma tissues and to investigate the effect of miRNA-99b on the cell invasion ability of hu-man glioma cell line U251. Methods The expres-sions of miRNA-99b and mTOR mRNA in glioma tis-sues and normal brain tissues were detected by real-time PCR. After co-transfection with miRNA-99b mim-ics and wild or mutation type mTOR 3′UTR recombina-tion vector,the specific binding ability of miRNA-99b to 3′-UTR in mTOR gene was examined by luciferase gene reporter system. The expression levels of miRNA-99b,mTOR mRNA and mTOR protein in glioma cell line U251 after transfected with miRNA-99b mimics were measured by real-time PCR and Western blot,re-spectively. The cell invasion was measured by Tran-swell assay. The changes of mTOR and miRNA-99b expression levels in U251 cells after transfection with mTOR PsiCHECK were detected by real-time PCR. The correlation between the expression of miRNA-99b and prognosis was analyzed statistically. Results The expressed level of miRNA-99b was lower, and the ex-pression level of mTOR was higher in the glioma tissues than that in the normal brain tissues. The expression of miRNA-99b was up-regulated, and the expressions of mTOR mRNA and protein were down-regulated in U251 cells after transfection with miRNA-99b mimics. However, the abilities of invasion of U251 cells after transfection with miRNA-99b mimics were inhibited. The relative protein expression levels of mTOR in mTOR PsiCHECK group were significantly different from those in negative control group, but the relative expression levels of miRNA-99b had no signifi-cant difference compared with those in negative control group. Over-expression of mTOR restored the abilities of cell invasion in U251 cells, which was reduced by miRNA-99b. The Kaplan—Meier analysis and Log-Rank Test showed that there were significant differ-ences in overall survival (OS) between the miRNA-99b high-expression and low-expression group. Con-clusions The expression level of miRNA-99b is low in human glioma tissue. miRNA-99b may inhibit the cell invasion activity of glioma cell line U251 in vitro via inhibiting mTOR expression.