BACKGROUND: Toll-like receptors (TLRs) are well-known pattern recognition receptors. Among the 13 TLRs, TLR2 is the most known receptor for immune response. It activates mitogen-activated protein kinases (MAPKs), which are counterbalanced by MAPK phosphatases [MKPs or dual-specificity phosphatases (DUSPs)]. However, the regulatory mechanism of DUSPs is still unclear. In this study, the effect of a TLR2 ligand (TLR2L, Pam3CSK4) on DUSP4 expression in Raw264.7 cells was demonstrated. METHODS: A Raw264.7 mouse macrophage cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at 37degrees C in 5% CO2. TLR2L (Pam3CSK4)-mediated DUSP4 expressions were confirmed with RT-PCR and western blot analysis. In addition, the detection of reactive oxygen species (ROS) was measured with lucigenin assay. RESULTS: Pam3CSK4 induced the expression of DUSP1, 2, 4, 5 and 16. The DUSP4 expression was also increased by TLR4 and 9 agonists (lipopolysaccharide and CpG ODN, respectively). Pam3CSK4 also induced ERK1/2 phosphorylation and ROS production, and the Pam3CSK4-induced DUSP4 expression was decreased by ERK1/2 (U0126) and ROS (DPI) inhibitors. U0126 suppressed the ROS production by Pam3CSK4. CONCLUSION: Pam3CSK4-mediated DUSP4 expression is regulated by ERK1/2 and ROS. This finding suggests the physiological importance of DUSP4 in TLR2-mediated immune response.
Acridines ; Animals ; Anti-Bacterial Agents ; Blotting, Western ; Butadienes ; Cell Line ; Dual-Specificity Phosphatases ; Macrophages ; Mice ; Mitogen-Activated Protein Kinase Phosphatases ; Mitogen-Activated Protein Kinases ; Nitriles ; Penicillins ; Phosphorylation ; Phosphotransferases ; Reactive Oxygen Species ; Receptors, Pattern Recognition ; Toll-Like Receptors

BACKGROUND: Aim of study is designed to investigate whether apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) expression is changed in abdominal aortic coarctation models. METHODS: Male Sprague-Dawley rats were randomly assigned with abdominal aortic coarctation, repaired group, sham, and control groups. Endothelial function was assessed with endothelium-dependent relaxations. Detection of superoxide anion and lipid peroxidation was performed by lucigenin chemiluminescence and thiobarbituric acid-reactive substances assay. APE1/Ref-1 expression was measured with Western blot and immunohistochemistry. RESULTS: In anesthetized condition, the abdominal aortic coarctation rats showed hypertension as systolic/diastolic arterial pressure of 171/114 mm Hg, compared with 114/94 mm Hg of control. Endothelium-dependent relaxations were significantly impaired in the aortic coarctation which was recovered in 1 week after coarctation repair. Superoxide production and lipid peroxidation were elevated in aortic coarctation rats. In immunohistochemistry, APE1/Ref-1 expressions were increased at aorta and kidney in aortic coarctation rats. Increased APE1/Ref-1 expression in aorta was recovered by repair of coarctation. CONCLUSIONS: Taken together, it suggests that APE1/Ref-1 expression was increased in aortic coarctation-induced hypertensive rats, suggesting a biomarker for hypertension. Impaired endothelium dependent relaxation in the aortic coarctation can be modulated by repair of coarctation or the modulation of blood pressure.
Acridines ; Animals ; Aorta ; Aortic Coarctation ; Arterial Pressure ; Blood Pressure ; Blotting, Western ; Endothelium ; Humans ; Hypertension ; Immunohistochemistry ; Kidney ; Lipid Peroxidation ; Luminescence ; Male ; Oxidation-Reduction ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Relaxation ; Salicylamides ; Superoxides

To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PKI cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP's inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Acridines ; pharmacology ; Animals ; Benzimidazoles ; metabolism ; Cell Cycle ; Cell Proliferation ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Genetic Vectors ; LLC-PK1 Cells ; cytology ; metabolism ; Mitoxantrone ; pharmacology ; Neoplasm Proteins ; genetics ; metabolism ; Plasmids ; Swine ; Tetrahydroisoquinolines ; pharmacology ; Transfection

Gastrodin (GAS) is the major bioactive component of the extracts from the rhizome of Gastrodia elata Blume. The aim of this study is to investigate the transport of GAS in Caco-2 cells and the interaction of P-glycoprotein and GAS. The apparent permeability coefficients (Papp) of GAS were measured as a function of directions and concentrations. It was demonstrated that the efflux ratio was < 2.0 over the range of 50-500 micromol x L(-1) of GAS from bi-directional transport studies. The transport rate of GAS was dependent on the concentrations. Papp of GAS was not affected by transport directions, GAS concentration or the classical inhibitors of P-glycoprotein (verapamil and GF 120918). The cellular accumulation of GAS in Bcap37/MDR1 cells transected with hMDR1 gene, was similar to that in Bcap37 cells. The accumulation in both cell lines was concentration dependent. GAS did not affect the accumulation of Rhodamine 123 in Bcap37/MDR1 cells over the range of 50-500 micromol x L(-1). It indicated that the transport of GAS in Caco-2 cell monolayers mainly is by passive paracellular transport pathway. P-glycoprotein did not participate in the absorption of GAS in the intestine or the transport across the blood-brain barrier.
ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; metabolism ; Acridines ; pharmacology ; Benzyl Alcohols ; isolation & purification ; pharmacokinetics ; Biological Transport ; Blood-Brain Barrier ; Breast Neoplasms ; pathology ; Caco-2 Cells ; Cell Line, Tumor ; Drug Resistance, Multiple ; Gastrodia ; chemistry ; Glucosides ; isolation & purification ; pharmacokinetics ; Humans ; Plants, Medicinal ; chemistry ; Rhodamine 123 ; pharmacokinetics ; Tetrahydroisoquinolines ; pharmacology ; Verapamil ; pharmacology

AIMTo study the effects of 9-(4-ethoxycarbonylphenoxy)-6,7-dimethoxy-1,2,3,4-tetrahydro acridine (EDT) on free radical induced injury in primary cultured rat cortical neuron and cerebral ischemia in mice.

METHODSIn primary rat cortical neuron, free radical injury model was established by 10 mumol.L-1 H2O2. The content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) in cells were investigated. Chronic cerebral ischemia model was produced by occlusion of one carotid artery and pneumogastric nerve in mice. The step down test was adopted to investigate the effect of EDT on the memory impairment. The cerebra morphology and MDA, NO content and SOD activity in mice cerebra were detected.

RESULTSIn primary rat cortical culture, 0.01-3 mumol.L-1 EDT concentration-dependently inhibited the formation of MDA and reduction of SOD activity induced by 10 mumol.L-1 H2O2. In chronic cerebral ischemia, EDT 2.5, 5 and 10 mg.kg-1 ig for 5 d greatly improved the memory impairment, reduced NO efflux and MDA content, while increased SOD activity in mice cerebra.

CONCLUSIONEDT was found to protect neurons from H2O2-induced neurotoxicity and inhibit chronic cerebral ischemia mediated injury and memory impairment in mice.

Acridines ; pharmacology ; therapeutic use ; Animals ; Animals, Newborn ; Brain Ischemia ; complications ; etiology ; prevention & control ; Cells, Cultured ; Cerebral Cortex ; cytology ; Female ; Hydrogen Peroxide ; antagonists & inhibitors ; Male ; Malondialdehyde ; metabolism ; Mice ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology ; therapeutic use ; Nitric Oxide ; metabolism ; Rats ; Rats, Wistar ; Superoxide Dismutase ; antagonists & inhibitors ; metabolism

In order to perform standardization in analytic methods of cryopreserved human semen, to investigate the differences of resistance to cryoinjury, to define the stage of critical cryoinjury during cryopreservation and to evaluate the quality change after thawing by time interval, the reviability of 30 normal and 30 abnormal semen were evaluated by the supravital stainings of spermatozoa using acridine orange and eosin yellow and the motility assay simultaneously according to the stages of freezing-thawing and the time interval after thawing. The results were summarized as follows: 1. Vitality was estimated higher than motility at all specimens and the gap between two became greater as motility decreased. 2. Reviability of abnormal semen was estimated lower than that of normal semen(p<0.05). 3. The critical cryoinjury to spermatozoa was noticed at the stage of freezing from 4 degrees C to -10 degrees C (p<0.05). 4. The significant decrease in quality of normal cryopreserved semen was noticed between 30 to 60 min. after thawing (p<0.05). These results suggest that the cryoinjury to human semen is different in nature, therefore it is advisable that the quality of cryopreserved human semen should be evaluated by vitality and motility assay simultaneously. And the resistance of abnormal semen to cryopreservation is so low that it would be difficult to be used clinically with satisfaction. Moreover the laboratory studies should be concentrated on freezing method to achieve better reviability and it is desirable in practice that post-thaw semen should be used within 30 min, after thawing.
Acridine Orange ; Cryopreservation ; Eosine Yellowish-(YS) ; Freezing ; Humans* ; Semen ; Spermatozoa*

A new technique of immediate sperm clearance has been developed by killing and irrigating residual sperm in the distal ductal system following vasectomy with a spermicidal solution at the time of the operation. For this, biochemical study, histologic study, and clinical study were conducted ,and by this, the need for postoperative contraception, and the need for postoperative semen testing were eliminated. Among the various spermicidal solutions such as tap water, distilled water,KMnO4,acetic acid, lactic acid, rivanol, alcohol, the KMnO4 solution has been proved to be the most suitable for the spermicidal solution, through biochemical study and histologic study. About 10ml of the KMnO4 solution,0.5% were injected into the distal ductal system under pressure, and killed redisual sperm immediately after the application. Number of redisual sperm in the distal ductal systems was significantly reduced by the KMnO4 application. No complication nor harmful effects could be observed after the procedure.
Contraception ; Ethacridine ; Homicide ; Lactic Acid ; Semen ; Spermatozoa ; Sterilization* ; Vasectomy* ; Water

Ceramic-on-polyethylene components have better wear characteristics than metal-on-polyethylene components in total hip arthroplasty (THA), and thus, extensive wear resulting in penetration of the femoral head through the acetabular cup is rare after ceramic-on-polyethylene THA. However, several reports have been issued regarding catastrophic polyethylene failure in ceramic-on-polyethylene systems. Here, the authors report the first case of bilateral complete polyethylene wear failure with acetabular cup perforation after ceramic-on-polyethylene THA.
Arthroplasty ; Ceramics ; Head ; Hip ; Polyethylene ; Tacrine

Ceramic-on-polyethylene components have better wear characteristics than metal-on-polyethylene components in total hip arthroplasty (THA), and thus, extensive wear resulting in penetration of the femoral head through the acetabular cup is rare after ceramic-on-polyethylene THA. However, several reports have been issued regarding catastrophic polyethylene failure in ceramic-on-polyethylene systems. Here, the authors report the first case of bilateral complete polyethylene wear failure with acetabular cup perforation after ceramic-on-polyethylene THA.
Arthroplasty ; Ceramics ; Head ; Hip ; Polyethylene ; Tacrine

For several centuries, orthopaedic surgeons have been given the task of managing the pain, limp, and gait disturbance that develop in association with various trauma and diseases of the hip joint. Progress has been made in development of surgical methods for the hip joint, and a recent concept was determined by introduction of the 'Clean Air Operating Enclosure' system and 'low frictional arthroplasty' in total hip arthroplasty (THA) by John Charnley, in 1960. THA has been performed as an exclusive method for hip joint reconstruction. We report on understanding of the current status from the past, present, and future trends with an introduction of the prior method of THA and a discussion of the history.
Arthroplasty ; Friction ; Gait ; Hip ; Hip Joint ; Tacrine