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

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

This study was aimed to assess the effects of fluid shear stress on the bone resorption in rat osteoclasts. The osteoclasts were derived from the lumbar vertebrae marrow cells which were isolated from the 6-month-old female Sprague Dawley rats, cultured on the slide at 1 x 10(6) cell/ml, and induced with 1, 25 (OH)2 Dihydroxyvitamin D3. The slide containing osteoclasts was taken out on day 7 after culture and then put into the flow chamber. The loads of fluid shear stress applied to the osteoclasts were 5.97, 11.36, 16.08 and 20.54 dyne/cm2, respectively, for 30 minutes. The osteoclasts unloading fluid shear stress were used as control. The bone resorptive pits were studied by light microscopy and scanning electron microscopy. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was detected with ultraviolet spectrophotometry. The results showed that fluid shear stress can increase the activity of TRAP and significantly increase the number and area of bone resorptive pits made by osteoclasts,and the effect of fluid shear stress on the bone resorption of osteoclasts is the same as that on the activity of TRAP. The reaction of the osteoclasts to the fluid shear stress in this study also suggested that the bone resorption of osteoclasts be increased in a magnitude of fluid shear stress-dependent manner, and that the changes of TRAP activity be closely related to the changes of the number and area of resorptive pits of the osteoclasts.
Acid Phosphatase ; metabolism ; Animals ; Bone Resorption ; physiopathology ; Calcitriol ; pharmacology ; Cells, Cultured ; Female ; Isoenzymes ; metabolism ; Lumbar Vertebrae ; cytology ; Osteoclasts ; cytology ; enzymology ; physiology ; Rats ; Rats, Sprague-Dawley ; Shear Strength ; Tartrate-Resistant Acid Phosphatase

It is now well established that several G protein- coupled receptors can signal without agonist stimulation (constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the Gi/o-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.
Adenylate Cyclase/antagonists&inhibitors/genetics/metabolism ; Animals ; Binding, Competitive ; Bornanes/metabolism/*pharmacology ; COS Cells ; Cannabinoids/metabolism ; Cercopithecus aethiops ; Isoenzymes/antagonists&inhibitors/genetics/metabolism ; Pyrazoles/metabolism/*pharmacology ; Rats ; *Receptor, Cannabinoid, CB2 ; Receptors, Cannabinoid ; Receptors, Drug/agonists/*antagonists&inhibitors/genetics/metabolism ; Signal Transduction/drug effects/physiology ; Transfection

Antineoplastic Agents ; pharmacology ; Bile ; physiology ; Bile Duct Neoplasms ; metabolism ; pathology ; Bile Ducts, Intrahepatic ; Celecoxib ; Cell Division ; drug effects ; Cell Line, Tumor ; Cholangiocarcinoma ; metabolism ; pathology ; Cyclooxygenase 2 ; Dinoprostone ; metabolism ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; Pyrazoles ; RNA, Messenger ; genetics ; Sphincterotomy, Transduodenal ; adverse effects ; Sulfonamides ; pharmacology ; Up-Regulation

OBJECTIVETo study the effects of Chinese medicine compound on bone metabolism of weightlessness rats simulated by tail suspension.

METHODFifty Wistar rats were divided into 5 groups randomly: control group, model group, and low does, medium dose and high does treated group. The experiment period lasted 21 days. After the Chinese compound prescription and distilled water were orally given to treated groups, and control and model group for 7 days, respectively, the tail suspension experiment was performed for treated and model group, meanwhile administration of Chinese compound prescription and distilled water still lasted until the end of the experiment. Blood serum was collected for determination of alkaline phosphatase (ALP), bone gla protein (BGP), tartrate-resistant acid phosphatase (TRAP), femoral bone HOP. The changes of bone mineral density (BMD) of femoral bone and lumbar vertebra were observe.

RESULTCompared with control group, the ALP level of model group was markedly decreased (P < 0.05), no change of BGP, TRAP was not observed, the BMD of femoral bone and lumbar vertebra were decreased remarkably (P < 0.05), Compared with model group, the change of ALP level of treated groups was not significant for all treated groups, the BGP level and BMD for medium dose group were increased (P < 0.05), the TRAP level for medium dose and high does groups was decreased (P < 0.05)

CONCLUSIONThe Chinese compound prescription can improve the bone formation and prevent bone loss via inhibiting bone absorption and improving ossify, bone mineral deposition and mineralization as well as increasing BMD, which leads to prevention and treatment of bone loss.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; metabolism ; Animals ; Bone Density ; drug effects ; Bone and Bones ; drug effects ; metabolism ; physiology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Hindlimb Suspension ; Isoenzymes ; metabolism ; Male ; Osteocalcin ; metabolism ; Rats ; Rats, Wistar ; Tartrate-Resistant Acid Phosphatase ; Weightlessness Simulation

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Rats ; Protein Interaction Mapping ; Protein Biosynthesis/drug effects ; Protein Binding/drug effects ; Phosphoproteins/chemistry/*metabolism ; Phospholipase C gamma/antagonists & inhibitors/chemistry/*metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Peptides/chemistry/metabolism ; PC12 Cells ; Neurofilament Proteins/chemistry/*metabolism ; Nerve Growth Factor/pharmacology ; Molecular Weight ; Molecular Sequence Data ; Microtubules/metabolism ; Microscopy, Fluorescence ; Isoenzymes/metabolism/pharmacology/physiology ; Glutathione Transferase/metabolism ; Blotting, Far-Western ; Blood Proteins/chemistry/*metabolism ; Binding Sites ; Animals ; Amino Acid Sequence

Abstract Phospholipase D (PLD) plays an important role as an effector in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, PLD2 was reported as a necessary intermediate in preventing apoptosis induced by hydrogen peroxide or hypoxia in rat pheochromocytoma (PC12) cells. The data presented here show that both PLD isozymes, PLD1 and PLD2 are also required in attenuating glutamate-induced cell death in PC12 cells. Treatment of PC12 cells with glutamate resulted in induction of apoptosis in these cells, which is accompanied by decreased PLD activity and increased ceramide concentration. Incubation of PC12 cells with exogenous C6-ceramide showed a time-dependent decrease of PLD activity. When cDNAs of PLD1 and PLD2 were transfected into PC12 cells respectively, overexpression of PLD1 or PLD2 resulted in inhibition of glutamate-induced apoptotic cell death. These data indicate that both PLD1 and PLD2 play a protective role against glutamate-induced cell death in PC12 cells.
Animals ; Apoptosis/drug effects/*physiology ; Cell Survival/drug effects ; Ceramides/pharmacology ; Dose-Response Relationship, Drug ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glutamic Acid/*toxicity ; Isoenzymes/drug effects/genetics/*metabolism ; Kinetics ; PC12 Cells ; Phospholipase D/chemistry/drug effects/genetics/*metabolism ; Rats ; Sphingolipids/metabolism

AIMTo determine how SDF-1 alpha/CXCR4 activates nuclear factor-kappa B (NF-kappaB) and promotes oral squamous cell carcinoma (OSCC) invasion.

METHODOLOGYA lentivirus-based knockdown approach was utilized to deplete gene expression. NF-kappaB activation was evaluated by Western blot analysis and electrophoretic mobility shift (EMSA).

RESULTSWe show that the activation of NF-kappaB by CXCR4 occurs through the Carma3/Bcl10/Malt1 (CBM) complex in OSCC. We found that loss of components of the CBM complex in HNSCC can inhibit SDF-1 alpha induced phosphorylation and degradation of IkappaBalpha, while TNF alpha induced IKK activation remains unchanged. Further, we identified a role for novel and atypical, but not classical, PKCs in activating IKK through CXCR4. Importantly, inhibition of the CBM complex leads to a significant decrease in SDF-1 alpha mediated invasion of OSCC.

CONCLUSIONThe CBM complex plays a critical role in CXCR4-induced NF-kappaB activation in OSCC. Targeting molecular components of the NF-kappaB signaling pathway may provide an important therapeutic opportunity in controlling the progression and metastasis of OSCC mediated by SDF-1 alpha.

Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; physiology ; B-Cell CLL-Lymphoma 10 Protein ; CARD Signaling Adaptor Proteins ; antagonists & inhibitors ; physiology ; Carcinoma, Squamous Cell ; pathology ; Caspase Inhibitors ; Caspases ; physiology ; Cell Line, Tumor ; Chemokine CXCL12 ; antagonists & inhibitors ; physiology ; Enzyme Activation ; drug effects ; Gene Silencing ; Genetic Vectors ; genetics ; Humans ; I-kappa B Kinase ; drug effects ; I-kappa B Proteins ; metabolism ; Isoenzymes ; antagonists & inhibitors ; Lentivirus ; genetics ; Membrane Proteins ; antagonists & inhibitors ; physiology ; Mouth Neoplasms ; pathology ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; physiology ; Neoplasm Invasiveness ; Neoplasm Proteins ; antagonists & inhibitors ; physiology ; Phosphorylation ; Plasmids ; genetics ; Protein Kinase C ; antagonists & inhibitors ; Receptors, CXCR4 ; physiology ; Tumor Necrosis Factor-alpha ; pharmacology