OBJECTIVETo investigate the effect of glucagon-like peptide-1 (GLP-1) on glycolipid metabolism in 3T3-L1 adipocytes and explore the mechanism.

METHODS3T3-L1 adipocytes were treated with GLP-1, insulin, or both for 24 h, and Western blotting was used to analyze the expression levels of adipose triglyceride lipase (ATGL), glucose transporter type 4 (GLUT4), Akt1, Akt2 and phosphorylated Akt in the cells. Immunofluorescence was used to observe lipid content in 3T3-L1 adipocytes.

RESULTSAkt1 and Akt2 were not activated by insulin stimulation in 3T3-L1 adipocytes. Akt was phosphorylated by GLP-1 stimulation, which inhibited the expression of ATGL and increased the translocation of GLUT4 from the intracellular membranes to plasma membranes. These changes were more obvious under the synergistic effect of insulin in 3T3-L1 adipocytes.

CONCLUSIONGLP-1 decreases lipolysis by inhibiting the expression of ATGL and improves insulin resistance by increasing the translocation of GLUT4 in 3T3-L1 adipocytes.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Membrane ; metabolism ; Down-Regulation ; Drug Synergism ; Glucagon-Like Peptide 1 ; pharmacology ; Glucose Transporter Type 4 ; metabolism ; Insulin ; pharmacology ; Insulin Resistance ; Intracellular Membranes ; metabolism ; Lipase ; metabolism ; Mice ; Phosphorylation ; Protein Transport ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.
Vacuoles/*metabolism ; Two-Hybrid System Techniques ; Toxoplasma/metabolism/*pathogenicity ; Protozoan Proteins/*metabolism/secretion ; Proteins/*metabolism ; Organelles/metabolism ; Intracellular Membranes/*metabolism ; Humans ; Hela Cells ; Gene Library ; Cytoplasmic Granules ; Animals

OBJECTIVETo study the effects of total flavone of Abelmoschl Manihot L. Medic (TFA) on the function of platelets and to explore its mechanism.

METHODSRat models of artery-veins bypassing thrombus formation were used. The platelets of rabbits were collected. Platelet aggregation was induced by collagen and intracellular calcium ion concentration ([Ca(2+)]i) was assayed by Fura-2 method.

RESULTSTFA (25, 50, 100 mg/kg) significantly and dose-dependently reduced the weight of thrombus. TFA (0.025, 0.05, 0.1 mg/ml) possessed dose-dependant inhibitory effects on rabbits' platelet aggregation induced by collagen. TFA significantly reduced the resting and CaCl(2)-induced increase of free intracellular calcium concentration ([Ca(2+)]i) in rabbit platelet in vitro.

CONCLUSIONTFA has an antiplatelet effect via the inhibition on the influx of Ca(2+).

Animals ; Blood Platelets ; drug effects ; Calcium ; blood ; Calcium Channel Blockers ; administration & dosage ; pharmacology ; Calcium Chloride ; pharmacology ; Carotid Artery Thrombosis ; blood ; etiology ; Collagen ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Flavones ; administration & dosage ; pharmacology ; Glycosides ; administration & dosage ; pharmacology ; Intracellular Membranes ; metabolism ; Osmolar Concentration ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; administration & dosage ; pharmacology ; Platelet Function Tests ; Rabbits ; blood ; Rats ; Rats, Wistar

AIMTo estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding.

METHODSThe mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB).

RESULTSThe cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions.

CONCLUSIONThe cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

Apoptosis ; Blotting, Western ; Caspases ; metabolism ; Cold Temperature ; adverse effects ; Cryopreservation ; Humans ; Immunomagnetic Separation ; Intracellular Membranes ; enzymology ; physiology ; Male ; Microscopy, Fluorescence ; Spermatozoa ; enzymology ; physiology

OBJECTIVETo investigate the effects of nitric oxide on mitochondrial permeability and cytochrome C (cyt C) of human hepatocellular carcinoma cell lines.

METHODSNO-mediated apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 was investigated by flow cytometry. The growth and proliferation of human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 were evaluted by MTT assay. Mitochondrial transmembrane potential was analyzed by flow cytometry with double staining of Rh123 and PI, and cytoplasmid cyt C was measured by Western blot. The cells were preincubate with cyclosporin A or GSH synthesis blocker BSO to explore their effect on the results of the above experiments.

RESULTSNO donor sodium nitroprusside (SNP) induced apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 and resulted in the decrease of the mitochondrial transmembrane potential and the increase of the amount of cytoplasmid cyt C in time-dependent manner. Cyclosporin A (CsA) specific inhibitor of the mitochondrial permeability transition pore could partially prevent the decrease of delta psi m and the release of cyt C. In contrast, GSH synthesis blocker BSO promoted the decrease of delta psi m and the release of cyt C.

CONCLUSIONSNO may induce apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 by decreasing delta psi m, opening the mitochondrial permeability transition pore, and releasing the cyt C.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Intracellular Membranes ; drug effects ; metabolism ; physiology ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; metabolism ; Nitric Oxide ; pharmacology ; Nitric Oxide Donors ; pharmacology ; Permeability ; drug effects

The relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.
Androgens/*pharmacology ; Animals ; Arteries/drug effects ; Calcium/*metabolism ; Coronary Vessels/*drug effects ; Female ; Intracellular Membranes/*metabolism ; Male ; Osmolar Concentration ; Potassium Channels, Voltage-Gated/drug effects/*metabolism ; Rabbits ; Support, Non-U.S. Gov't ; Testosterone/*pharmacology ; *Vasodilation

The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non-specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM-treated cells, and FCM and RT-PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl-2 and mitochondrial transmembrane potential in AM-treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration-dependent manner. Morphologic observations showed apoptosis changes in AM-treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT-PCR detection showed that AM intervention increased the fraction of annexin V(+) cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down-regulated the expression of Bcl-2 at both mRNA and protein level and cyclin B1 protein in a concentration-dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down-regulation of Bcl-2 and reduction of mitochondrial transmembrane potential.
Aminophylline ; pharmacology ; Apoptosis ; drug effects ; Burkitt Lymphoma ; drug therapy ; genetics ; pathology ; Cell Division ; drug effects ; Cyclin B ; genetics ; metabolism ; Cyclin B1 ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Phosphodiesterase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; drug effects ; genetics ; metabolism ; S Phase ; Tumor Cells, Cultured ; drug effects ; metabolism ; ultrastructure

OBJECTIVETo probe the possible mechanism of growth-inhibitory and apoptosis of oral squamous cell carcinoma by arsenic trioxide.

METHODSThe induction of apoptosis in two tongue squamous carcinoma cells treated by arsenic trioxide was investigated. The morphology changes of the cells was observed under electron microscope. The mitochondrial transmembrane potential was detected using rhodamine 123 and flow cytometry. The cell cycle was detected by flow cytometry, and p16, p53, BCL-2, Caspase-3, and PARP changes were examined by western blot.

RESULTS1. The antiproliferative effect on the oral squamous carcinoma cells by arsenic trioxide was carried out through two ways: induction of apoptosis and toxicity damage. 2. Activation of the caspase-3 and PARP, while no changes of p16, p53, BCL-2 occurred. 3. Mitochondrial transmembrane potential collapse and G(2)-M stagnation were correlated with apoptosis of oral squamous cell carcinoma.

CONCLUSIONS1. Tubulins and mitochondria may be the chief action position of arsenic trioxide, which is the start-up factors of mechanism. 2. Activation of the caspase-3 proteolytic pathway may be one of the pivotal ways of apoptosis procedure induced by arsenic trioxide.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell ; drug therapy ; metabolism ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; DNA, Neoplasm ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Mouth Neoplasms ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Cells, Cultured ; drug effects ; ultrastructure ; Tumor Suppressor Protein p53 ; metabolism

In order to explore the differences between the mechanisms of selenite-induced apoptosis and arsenic induced apoptosis in NB4 cells, growth inhibition was determined by MTT test, apoptosis determined by DNA electrophoresis and analysis of intracellular DNA contents, reactive oxygen species and reduced glutathione in the cell were measured by Lucigenin dependent chemoluminescent (CL) test and spectrophotometry, and mitochondrial transmembrane potential was measured by flow cytometry. The results showed that: 5 micro mol/L sodium selenite similar to 1 micro mol/L arsenic trioxide could induce the apoptosis of NB4 cells after treatment for 24 hours. Both could elevate the level of reactive oxygen species and intensify mitochondrial transmembrane potential collapse, accompanied with decrease of reduced glutathione centent. The effect of selenium selenite on these aspects was more significant than those of arsenic trioxide. Elevation of intracellular glutathione in N-acytlcysteine pretreated NB4 cells could enhance the selenite induced apoptosis and oxidative stress, but ameliorate the arsenic trioxide induced apoptosis and oxidative stress. It was concluded that sodium selenite and arsenic trioxide can induce the apoptosis of NB4 cells, but there are significant differences between the mechanisms of selenite-induced and arsenic-induced apoptosis in NB4 cells, particularly in the influence of intracellular glutathione content on the drug action.
Acetylcysteine ; pharmacology ; Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; Cell Division ; drug effects ; DNA, Neoplasm ; drug effects ; genetics ; metabolism ; Flow Cytometry ; Glutathione ; drug effects ; metabolism ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Oxides ; pharmacology ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; Tumor Cells, Cultured ; drug effects

To elucidate the Ca2+ release mechanisms in the rabbit coronary artery, arterial preparations were permeabilized with beta-escin and changes in tension were measured under varying experimental conditions. Additionally, we investigated properties and distribution of two kinds of Ca2+ release mechanisms, Ca2+-induced Ca2+ release (CICR) and IP3-induced Ca2+ release (IICR). The results obtained were summarized as follows; 1. When a rabbit coronary artery was incubated in a relaxing solution containing 30 microM beta-escin for 40 min. sensitivity to externally added Ca2+ was much higher in beta-escin permeabilized muscle than in intact preparations. The contractile effect of IP3 in beta-escin permeabilized muscle was also demonstrated; 2. Caffeine and IP3 contracted coronary arteries were permeabilized with beta-escin, but the amplitude of contraction was much larger in the presence of caffeine than of IP3. 3. Intracellular heparin completely inhibited the contractions induced by IP3, but not those by caffeine. On the other hand, procaine inhibited the responses to caffeine, but not those to IP3. Ryanodine inhibited both the caffeine- and IP3-induced contractions. 4. The amplitude of contractile responses was much larger to the maximal stimulation of CICR by applying caffeine than to the maximal stimulation of IICR by applying IP3. After the maximal CICR stimulation by caffeine, the activation of IICR by IP3 without the reloading of Ca2+ could no longer evoke contraction. On the other hand, after the maximal IICR activation, the activation of CICR could still evoke contraction although the amplitude of the contraction was smaller when compared with the case without the initial IICR stimulation. 5. Acetylcholine contracted coronary artery smooth muscles were permeabilized with beta-escin. However, in the absence of added guanosine triphosphate (GTP), the responses were very small. Acetylcholine-induced contraction was inhibited by heparin, but not by procaine. From the above results, it may be concluded that there are two kinds of mechanisms of Ca2+ release, CICR and IICR, in the rabbit coronary artery smooth muscle cell. Also, whereas the CICR mechanism distributes on the membrane of the whole smooth muscle Ca2+ store, the IICR mechanism distributes only on a part of it.
Animal ; Arteries/metabolism ; Calcium/*metabolism ; Capillary Permeability/drug effects ; Coronary Vessels/drug effects/*metabolism ; Escin/pharmacology ; In Vitro ; Intracellular Membranes/*metabolism ; Rabbits ; Support, Non-U.S. Gov't ; Tissue Distribution