Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.
Animal ; Biological Transport/drug effects ; Dexamethasone/pharmacology* ; Dose-Response Relationship, Drug ; Drug Interactions ; Glycerophospholipids/analysis ; Isoenzymes/drug effects ; Kidney Cortex/cytology ; Kidney Tubules, Collecting/drug effects* ; Kidney Tubules, Collecting/cytology ; Mice ; Mice, Transgenic ; Oleic Acid/pharmacology ; Phospholipase D/drug effects*

OBJECTIVETo investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G bacteria, E. coli K12 and Pse.FH2, and one G+ bacterum, B. subtilis.

METHODSThe SOD, CAT, and ATPase specific activities of cell lysates were determined spectrophotometrically at 550 nm, 240 nm, and 660 nm, respectively, with kits A001, A016, and A007. SOD isozyme patterns were detected by native PAGE analysis.

RESULTSSOD and CAT activities in the tested bacteria increased significantly in a concentration-dependent manner after different concentrations of acetamiprid were applied. The activity of SOD in B. subtilis and Pse.FH2 was stimulated and reached the highest level after treatment with 100 mg/L acetamiprid for 0.5 h. For Pse.FH2, there was another stimulation of SOD activity after acetamiprid application for about 8.0 h and the second stimulation was stronger than the first. The stimulation by acetamiprid showed a relative lag for E. coli K12. Acetamiprid seemed to exhibit a similar effect on CAT activity of the two G bacteria and had an evident influence on ATPase activity in the three bacteria within a relatively short period. Only one SOD isozyme was detectable in Pse.FH2 and B. subtilis, while different isozyme compositions in E. coli could be detected by native PAGE analysis.

CONCLUSIONAcetamiprid causes a certain oxidative stress on the three bacteria which may not only elevate SOD and CAT activities but also generate new SOD isozymes to antagonize oxidative stress. However, this oxidative stress lasts for a relatively short time and does not cause a long-term damage.

Adenosine Triphosphatases ; metabolism ; Bacillus ; drug effects ; enzymology ; Bacteria ; drug effects ; enzymology ; Catalase ; metabolism ; Escherichia coli ; drug effects ; enzymology ; Insecticides ; pharmacology ; Isoenzymes ; metabolism ; Neonicotinoids ; Pseudomonas ; drug effects ; enzymology ; Pyridines ; pharmacology ; Superoxide Dismutase ; metabolism

AIMTo evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis.

METHODSFor creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA.

RESULTSA significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1, whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1.

CONCLUSIONExcessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level.

Animals ; Diet ; Glutathione Transferase ; metabolism ; Isoenzymes ; drug effects ; metabolism ; L-Lactate Dehydrogenase ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; drug effects ; physiology ; Selenium ; deficiency ; pharmacokinetics ; pharmacology ; Spermatogenesis ; physiology ; Testis ; drug effects ; enzymology ; pathology ; physiology

OBJECTIVETo investigate the expressions of protein kinase C (PKC) isoforms in X-ray-exposed HepG2 cells and identify the PKC isoforms that induce radioresistance in HepG2 cells.

METHODSCultured HepG2 cells were divided into control group and 6 Gy radiation group for corresponding treatments. The fluorescence intensity (FI) and the percentage of positive cells were determined using flow cytometry.

RESULTSThe FI of PKCalpha and PKCdelta were 2.28 and 5.05 in the radiation group, respectively, significantly higher than those in the control group (P<0.05). The percentages of PKCalpha- and PKCdelta -positive cells were significantly higher in the radiation group than in the control group (P<0.05). The FI and the percentages of PKC zeta, gamma, epsilon, zeta positive cells were rather low and showed no significant differences between the two groups (P>0.05); PKCbeta expression was not detected in the two groups of cells. The apoptosis rates of the control and radiation groups were 1.73% and 20.90%, respectively.

CONCLUSIONPKCalpha and PKCdelta may be involved in protecting HepG2 cells from radiation-induced apoptosis.

Apoptosis ; physiology ; radiation effects ; Hep G2 Cells ; Humans ; Isoenzymes ; classification ; metabolism ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-delta ; metabolism ; Radiation Tolerance ; Signal Transduction ; drug effects ; physiology

Methyl-beta-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-beta-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-beta-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.
Animals ; Cell Cycle/drug effects/*physiology ; Cell Line ; Cell Proliferation/*drug effects ; Dinoprostone/*metabolism ; Dose-Response Relationship, Drug ; Isoenzymes/genetics/*metabolism ; Macrophages/cytology/*drug effects/physiology ; Mice ; Prostaglandin-Endoperoxide Synthase/genetics/*metabolism ; Research Support, Non-U.S. Gov't ; beta-Cyclodextrins/*pharmacology

OBJECTIVETo compare the ability of osthole (OST) and genistein (GEN) in enhancing bone peak bone mass of rats to prevent osteoporosis.

METHODSThirty-six female one-month-old SD rats of (125 +/- 3) g body weight were randomly divided into three groups, 12 rats in each group, one group was orally administered osthole at 9 mg x kg(-1) d(-1), one group was given genistein at 10 mg x kg(-1) d(-1) and another was given equal quantity of distilled water as the control. The body weight was monitored weekly and the bone mineral density (BMD) of total body was measured every month. All rats were sacrificed after three months, the femoral bone mineral density, the serum levels of osteocalcin (OC) and anti-tartaric acid phosphatase 5b (TRACP 5b) were measured by Elisa. The bone microarchitectures were analyzed with micro-CT and the bone biomechanics properties were tested with universal material machine.

RESULTSNo significant differences were observed between O-treated or GEN group and the control for the food-intake and body weight during three months. However, the rats treated with OST had significant higher BMD for both total body and femur than the control and GEN group. The O-treated rats also had higher level of serum OC and lower level of TRACP 5b. Besides, they owned bigger bone volume/tissue volume, trabecular thickness, trabecular number but smaller trabecular spacing. In the three point bending tests of femurs,they were found to have larger maximum load, the young's modulus and structural model index (SMI).

CONCLUSIONOrally administered osthole could efficiently increase the peak bone mass of rats,which provide new ideas for preventing osteoporosis.

Acid Phosphatase ; blood ; Animals ; Body Weight ; drug effects ; Bone Density ; drug effects ; Coumarins ; pharmacology ; Female ; Femur ; diagnostic imaging ; drug effects ; pathology ; Genistein ; pharmacology ; Isoenzymes ; blood ; Osteocalcin ; blood ; Radiography ; Rats ; Rats, Sprague-Dawley ; Tartrate-Resistant Acid Phosphatase

OBJECTIVETo discuss the effect of Drynariae Rhizoma's naringin on osteoclasts induced by mouse monocyte RAW264.7.

METHODRAW264.7 cells were induced by 100 μg x L(-1) nuclear factor-κB receptor activator ligand (RANKL) and became mature osteoclasts, which were identified through TRAP specific staining and bone resorption. MTT method was sued to screen and inhibit and the highest concentration of osteoclasts. After being cultured with the screened medium containing naringin for 5 days, positive TRAP cell counting and bone absorption area analysis were adopted to observe the effect of naringin on the formation of osteoclast sells and the bone absorption function. The osteoclast proliferation was measured by flow cytometry. The effects of RANK, TRAP, MMP-9, NFATc1 and C-fos mRNA expressions on nuclear factor-κB were detected by RT-PCR.

RESULTNaringin could inhibit osteoclast differentiation, bone absorption function and proliferation activity of osteoclasts, significantly down-regulate RANK, TRAP, MMP-9 and NFATc1 mRNA expressions in the osteoclast differentiation process, and up-regulate the C-fos mRNA expression.

CONCLUSIONNaringin could inhibit osteoclast differentiation, proliferation and bone absorption function. Its mechanism may be achieved by inhibiting the specific gene expression during the osteoclast differentiation process.

Acid Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flavanones ; pharmacology ; Isoenzymes ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Mice ; NFATC Transcription Factors ; genetics ; Osteoclasts ; cytology ; drug effects ; Tartrate-Resistant Acid Phosphatase

OBJECTIVETo evaluate the roles or effects of oviductus ranae (OR) or oviductus ranae eggs (ORE) in preventing and treating postmenopausal osteoporosis.

METHODSIn vivo experiment: Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized (OVX), with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens (CE), OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein (BGP), serum calcium, phosphorus, and alkaline phosphatase (ALP) were assayed; bone mineral densities (BMD) were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment: Eighty 10-month old SD rats, with equal numbers of males and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase (TRAP).

RESULTSIn vivo experiment: The body weight of the four OVX groups increased significantly (P<0.01). Uterine weight of the CE group was the highest (P<0.01); Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly (P<0.05, P<0.01); femoral volume in the ORE group increased significantly (P<0.05); and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly (P<0.05, P<0.01). In vitro experiment: Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts (P<0.05 and P<0.01, repsectively) and stimulated the formation of calcium nodes (P<0.05, P<0.01), while the enhancement of ALP activity in osteoblasts was significant (P<0.05, P<0.01). The number of TRAP-positive cells was significantly reduced as well (P<0.01).

CONCLUSIONSOR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; metabolism ; Animals ; Biomarkers ; blood ; Body Weight ; drug effects ; Bone Density ; drug effects ; Bone and Bones ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Female ; Femur ; drug effects ; metabolism ; pathology ; Isoenzymes ; metabolism ; Male ; Materia Medica ; pharmacology ; therapeutic use ; Organ Size ; drug effects ; Osteoblasts ; drug effects ; enzymology ; pathology ; Osteoclasts ; drug effects ; enzymology ; pathology ; Osteoporosis ; blood ; drug therapy ; metabolism ; physiopathology ; Ovariectomy ; Ovum ; metabolism ; Rats ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase ; Uterus ; drug effects ; pathology

To research the influence of Reduning injection on the activity and mRNA expression of cytochrome P450 (CYP450) system in rat liver microsomes. Rat liver microsomes were prepared after a seven-days continuous administration of Reduning injection. An HPLC-MS method was applied to determine the specific metabolites of CYP450 probe substrates in rat liver microsomal incubations. The activity of CYP450 isozymes were represented by the formation of metabolites. The Real-time quantitative polymerase chain reaction (Q-PCR) was applied to determine the mRNA expression levels of CYP450. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13 (P < 0.01), but did not affect CYPlA2; low dose and high dose of Reduning injection had an inhibition trend on the activity of CYP2D2, but did not statistically differ from control group; low dose of Reduning injection significantly induced the activity of CYP3A1 (P < 0.01), high dose of Reduning injection had an induce trend on the activity of CYP3A1, but did not statistically differ from control. At the mRNA level, low and high dose of Reduning injection had an induce trend on the expression of CYP1A2, 2C11, 2D1, 2E1, 3A1, but did not statistically differ from control. Reduning injection significantly induced the activity of CYP2B1. Reduning injection significantly induced the activity of CYP3A1 in mRNA expression and enzyme activity levels, which may result adverse drug reaction after being combined with macrolides antibiotics. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13, 2D2 in enzyme activity levels, when combined with other drugs, it should be fully taken into account of the possible drug-drug interaction in order to avoid adverse side effects.
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Injections ; Isoenzymes ; metabolism ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the molluscicidal activities of three Chinese plants N. indicum Mill, R stenoptera DC, and R. japonicum Houtt, and to clarify the molluscicidal mechanism.

METHODSN-butanol extracts and water extracts of the three plants were obtained. The reactions of EST isozyme, glycogen and total protein of snails to the plant extracts were studied.

RESULTSEST electrophoresis showed that EST was an important antidotal enzyme system and reacted strongly to environment. EST changed greatly during the whole exposure period so that it could be viewed as a pathological index of toxicity. Extracts decreased the glycogen content of the snails' soft tissues greatly, and also the protein content.

CONCLUSIONAll extracts show strong molluscicidal activity. The LD50 value of the water extract of N. indicum Mill is as low as 13.2 mg/L. EST can be viewed as a pathological index of toxicity. The energy metabolism abnormity is the key reason for the molluscicidal activities. The biochemical mechanism needs further research.

Animals ; Electrophoresis, Polyacrylamide Gel ; Esterases ; metabolism ; Glycogen ; metabolism ; Isoenzymes ; metabolism ; Juglandaceae ; chemistry ; toxicity ; Molluscacides ; toxicity ; Nerium ; chemistry ; toxicity ; Plant Extracts ; chemistry ; toxicity ; Rumex ; chemistry ; toxicity ; Snails ; drug effects