OBJECTIVETo investigate the effects of Tongfeng trace elements nutrient balance agent on the various growth indicators, physiological indicators, and the contents of liquiritin and glycyrrhizic acid in one-year old Glycyrrhiza uralensis.

METHODThe plants of G. uralensis growing in Chifeng of Inner Mongolia and medicinal garden of Beijing University of Chinese Medicine were fertilized for two times, respectively. The photosynthetic physiological indicators were measured by LI-6400 photosynthetic instrument. The pigments and antioxidase activities of the leaves were determined. Then contents of liquiritin and glycyrrhizic acid in the plants were determined by HPLC.

RESULTThe application of this trace element nutrient balance agent could significantly improve the height, chla and chlb, and the photosynthetic physiology indicator such as P(n), C(i), and G(s). Similarly, it could significantly increase the fresh weight of shoots and dry weight of the roots. Compared with control block (CK), the fertilizer which was diluted by 300 times (T(1)) and 600 times (T(2)) significantly increased the content of glycyrrhizic acid by 24.72% and 20. 23%. There was significant difference between different treatments (P < 0.05).

CONCLUSIONThe Tongfeng trace elements nutrient balance agent could promote growth, physiology and the content of active constituents of G. uralensis, especially the effect of T(1) was superior to T(2).

Enzyme Activation ; drug effects ; Fertilizers ; Flavanones ; metabolism ; Glucosides ; metabolism ; Glycyrrhiza uralensis ; drug effects ; growth & development ; physiology ; Glycyrrhizic Acid ; metabolism ; Oxidoreductases ; metabolism ; Photosynthesis ; drug effects ; Trace Elements ; pharmacology

OBJECTIVETo investigate the effect of telomerase activation on biological behaviors of neural stem cells after hypoxic-ischemic insults.

METHODSThe neural stem cells passaged in vitro were divided into four groups: control, oxygen-glucose deprivation (OGD), OGD+cycloastragenol (CAG) high concentration (final concentration of 25 μM), and OGD+CAG low concentration (final concentration of 10 μM). The latter three groups were subjected to OGD. Telomerase reverse transcriptase (TERT) expression level was evaluated by Western blot. Telomerase activity was detected by telomerase repeat amplification protocol (TRAP). Cell number and neural sphere diameter were measured under a microscope. The activity of lactate dehydrogenase (LDH) was examined by chemiluminescence. Cell proliferation rate and apoptosis were detected by flow cytometry.

RESULTSAfter OGD insults, obvious injury of neural stem cells was observed, including less cell number, smaller neural sphere, more dead cells, lower proliferation rate and decreased survival rate. In CAG-treated groups, there were higher TERT expression level and telomerase activity compared with the control group (P<0.05). In comparison with the OGD group, CAG treatment attenuated cell loss (P<0.05) and neural sphere diameter decrease (P<0.05), promoted cell proliferation (P<0.05), and increased cell survival rate (P<0.05). Low and high concentrations of CAG had similar effects on proliferation and survival of neural stem cells (P>0.05). In the normal cultural condition, CAG treatment also enhanced TERT expression (P<0.05) and increased cell numbers (P<0.05) and neural sphere diameter (P<0.05) compared with the control group.

CONCLUSIONSTelomerase activation can promote the proliferation and improve survival of neural stem cells under the state of hypoxic-ischemic insults, suggesting telomerase activators might be potential agents for the therapy of hypoxic-ischemic brain injury.

Animals ; Cell Survival ; drug effects ; Enzyme Activation ; Hypoxia-Ischemia, Brain ; etiology ; Neural Stem Cells ; drug effects ; physiology ; Rats ; Sapogenins ; pharmacology ; Telomerase ; physiology

The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.
Apoptosis ; Caspases/metabolism ; Catalase/pharmacology ; Cell Line ; Cell Survival/drug effects ; Enzyme Activation ; Human ; Hydrogen Peroxide/*pharmacology ; Necrosis ; Pigment Epithelium of Eye/*drug effects/enzymology/pathology/*physiology

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0 approximately10 microg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10(-8) to 10(-7) mol/L and the PKC inhibitor H(7) inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H(7). These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.
Animals ; Cell Proliferation ; drug effects ; Enzyme Activation ; Ginsenosides ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Proliferating Cell Nuclear Antigen ; analysis ; Protein Kinase C ; physiology ; Spermatogonia ; cytology ; drug effects

OBJECTIVETo study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.

METHODSVero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy.

RESULTIn enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells.

CONCLUSIONThe results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.

Animals ; Cell Nucleus ; physiology ; Cercopithecus aethiops ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA Damage ; Enzyme Activation ; drug effects ; Methylnitronitrosoguanidine ; toxicity ; Receptor, Epidermal Growth Factor ; drug effects ; metabolism ; Receptors, Tumor Necrosis Factor ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Vero Cells

Normal human epidermal keratinocytes (NHEK) respond to the autocrine activated extracellular signal-regulated kinase (ERK) signaling pathway, which contributes to the survival of keratinocytes. However, during the condition of calcium-induced differentiation, how the autocrine ERK signaling is regulated and affected is poorly understood. The purpose of this study was to understand and to obtain clues to the possible function of the autocrine ERK activation during the calcium-induced differentiation of NHEK. We demonstrated that the autocrine activated ERK was not interrupted by calcium triggering and that it was sustained for at least one day after changing the medium. We also found that the autocrine ERK activation was associated with the expression of cyclin D1 and the cell cycle regulation at the early stage of calcium triggering by treating the cells with the mitogen-activated protein kinase inhibitor PD98059. However, the PD98059 treatment did not have a significant influence on the expression of involucrin and loricrin. In addition, we demonstrated that autocrine ERK activation was associated with protein kinase C and p38MAPK signaling. We suggest that the activation of autocrine ERK is not interrupted by calcium triggering and it might participate in cell growth during the early stage of calcium-induced differentiation in NHEK.
Keratinocytes/*cytology/drug effects/*physiology ; Humans ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Enzyme Activation/drug effects ; Dose-Response Relationship, Drug ; Cells, Cultured ; Cell Differentiation/drug effects ; Cell Cycle/drug effects/*physiology ; Calcium Signaling/drug effects/*physiology ; Calcium/*administration & dosage ; Autocrine Communication/drug effects/*physiology

BACKGROUNDWe have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.

METHODSWe assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.

RESULTSThe NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.

CONCLUSIONThese results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; DNA Fragmentation ; drug effects ; Enzyme Activation ; Humans ; Melanoma ; drug therapy ; pathology ; Mitogen-Activated Protein Kinases ; physiology ; Protein Kinase C ; antagonists & inhibitors ; physiology ; Staurosporine ; pharmacology

A modified electrometric method was described and validated for measurement of plasma and erythrocyte cholinesterase activities in 6~18 months old goats. The enzymatic reaction mixture contained 3 ml distilled water, 3 ml barbital-phosphate buffer (pH 8.1), 0.2 ml plasma or erythrocytes and 0.1 ml acetylthiocholine iodide (7.5%) as a substrate. The mixture was incubated at 37 degrees C for 40 minutes. The pH of the reaction mixture was determined by a pH meter before and after the incubation. The initial pH was measured before the substrate addition. The enzyme activity was expressed as deltapH/40 min. The coefficients of variation of the described method in measuring plasma and erythrocyte cholinesterase activities were 4 and 2%, respectively. Preliminary reference values (n = 14) of the mean cholinesterase activity (deltapH/40 min) and 95% confidence interval in the plasma were 0.194 and 0.184~ 0.204, respectively, and those of the erythrocytes were 0.416 and 0.396~0.436, respectively. The pseudocholinesterase activity of the plasma cholinesterase was 63.5% as determined by quinidine sulfate inhibition. The organophosphorus insecticides dichlorvos and diazinon at 0.5~4 micrometer and the carbamate insecticide carbaryl at 5~20 micrometer in the reaction mixture significantly inhibited plasma (13.7~85.5%) and erythrocyte (16.4~71.9%) cholinesterases in vitro in a concentration-dependent manner. The results suggest that the described electrometric method is simple, precise and efficient in measuring blood cholinesterase activity in goats.
Acid-Base Equilibrium/physiology ; Animals ; Carbaryl/pharmacology ; Cholinesterase Inhibitors/pharmacology ; Cholinesterases/*blood/drug effects ; Diazinon/pharmacology ; Dichlorvos/pharmacology ; Enzyme Activation/drug effects/physiology ; Erythrocytes/metabolism ; Goats/*blood ; Plasma/metabolism

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.
Animals ; Cell Movement/drug effects/*physiology ; Cells, Cultured ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Genistein/pharmacology ; Hydrogen Peroxide/pharmacology ; Muscle, Smooth, Vascular/cytology/*physiology ; *Oxidative Stress/drug effects ; Phospholipase D/*metabolism ; Phosphorylation/drug effects ; Protein Kinase C/*metabolism ; Protein-Tyrosine Kinase/antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Signal Transduction/drug effects ; Vanadates/pharmacology ; Vascular Diseases/metabolism

OBJECTIVETo investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) on multiple myeloma (MM) cell migration and adhesion and it related signaling pathways.

METHODSExpression of adhesion molecules on MM cells of RPMI8226, XG-1 and XG-7 cells was analysed by flow cytometry, the influence of SDF-1 on CD29 and CD49e distribution by immunofluorescence, the effect of SDF-1 on chemotaxis of MM cells by transwell assay. Activation of phosphoinositide-3 kinase (PI3K) in MM cells treated with SDF-1 and by immunoblotting.

RESULTS3 strains of MM cell line expressed many adhesion molecule. RPMI8226, XG-7 cells were all high level of expression of CD29 (> 70%). XG-1, XG-7 cells were all high level of expression of CD44 (> 80%), and XG-7 cells was of CD49d (> 90%). In all of 3 strains, the levels of expression of CD49e were low (< 30%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of MM cells and the redistribution of CD29 and CD49e. SDF-1 promoted MM cells adhesion to endothelial cells, stimulated phosphorylation of P85 subunit of PI3K in MM cells and induced MM cells migration, which were inhibited by G protein inhibitor PTX and PI3K inhibitor wortmannin.

CONCLUSIONSDF-1 can promote MM cell adhesion to endothelial cells, trigger establishment of a polarized morphology of MM cells and redistribution of adhesion molecules and induce MM cells migration via PI3K signaling pathway.

Blotting, Western ; Cell Adhesion ; drug effects ; physiology ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; physiology ; Chemokine CXCL12 ; pharmacology ; physiology ; Enzyme Activation ; drug effects ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans ; Integrin alpha4 ; metabolism ; Integrin alpha5 ; metabolism ; Integrin beta1 ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; physiopathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; drug effects ; physiology