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

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes have typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin. GSLs are usually observed in many organs ubiquitously. However, GSLs, including over 400 derivatives, participate in diverse cellular functions. Several studies indicate that GSLs might have an effect on signal transduction related to insulin receptors and epidermal growth factor receptors. GSLs may modulate immune responses by transmitting signals from the exterior to the interior of the cell. Guillain-Barre syndrome is one of the autoimmune disorders characterized by symmetrical weakness in the muscles of the legs. The targets of the immune response are thought to be gangliosides, which are one group of GSLs. Other GSLs may serve as second messengers in several signaling pathways that are important to cell survival or programmed cell death. In the search for clear evidence that GSLs may play critical roles in various biological functions, many researchers have made genetically engineered mice. Before the era of gene manipulation, spontaneous animal models or chemical-induced disease models were used.
Animals ; Caveolae ; Cell Death ; Cell Membrane ; Cell Survival ; Cholesterol ; Diabetes Mellitus ; Gangliosides ; Glycosphingolipids ; Guillain-Barre Syndrome ; Intracellular Membranes ; Leg ; Mice ; Models, Animal ; Muscles ; Receptor, Epidermal Growth Factor ; Receptor, Insulin ; Second Messenger Systems ; Signal Transduction ; Sphingolipids

PURPOSE: Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. The purposes of this study were to investigate the localization and functional roles of AQP-1 water channels in rat vagina. MATERIALS AND METHODS: Female Sprague-Dawley rats (230~240 g, n=40) were anesthetized. To investigate the expression and localization of AQPs in the vagina, the vaginal branch of the pelvic nerve was stimulated for 60 seconds (10 V, 16 Hz, 0.8 msec), and then the animals were sacrificed immediately or 5 minutes later. The expression and cellular localization of AQP-1 in the vaginal tissue was measured by Western blot and immunohistochemistry. The cytosolic (or intracellular membrane) and plasma membrane fractions of AQP-1 in vaginal tissue were studied by immunoblot analysis. RESULTS: Immunolabeling showed that AQP-1 was mainly expressed in the capillaries and venules of the vagina. The translocation of AQP-1 isoforms from the cytosolic compartment to the plasma membrane compartment was observed right after nerve stimulation and had declined at 5 minutes after nerve stimulation. However, when the nerve stimulation was repeated 3 times, the translocation of AQP-1 from the intracellular membrane compartment to the plasma membrane compartment was still observed at 5 minutes. CONCLUSIONS: This study suggests that sexual arousal induced by pelvic nerve stimulation modulates AQP-1 activity in the rat vagina. This result implies that AQP-1 may play an important role in vaginal lubrication.
Animals ; Aquaporins ; Arousal ; Blotting, Western ; Capillaries ; Cell Membrane ; Cytosol ; Female ; Humans ; Immunohistochemistry ; Intracellular Membranes ; Lubrication ; Membrane Proteins ; Membranes ; Protein Isoforms ; Rats* ; Rats, Sprague-Dawley ; Vagina* ; Venules ; Water Movements

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

Study on the peroxidation of membrane lipid and the change of some anti-oxidant enzymes during the storage of erythrocytes by additive system. The invitro study was conducted with two additive systems: one is the AS-T which is the first preparation in VN and the other is the preparation of Terumo, a leading company of the world on the blood preparations. The results demonstrate that the two systems are equally in the anti-peroxidation activation of membrance lipid. The ability to maintain enzyme activation of Superoxyd dismutase (SOD) and Gluathion peroxidase (GCH-Px) of erythrocytes during of storage process (from ) to 35 days), the erythrocyte mass which is preserved by AST solution abilize activity of SOD and GSH-Px better than by Terumo solution, with this result os signficant slower increasing of MDA concentration at the end of storage
Lipids ; Intracellular Membranes ; Erythrocytes

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

Japanese encephalitis virus (JEV) is one of the most important human pathogens, which causes the permanent neuropsychiatric sequelae and even fatal diseases with high mortality and morbidity, especially among children. In this study, we expressed the structural proteins (C, prM, and E) of JEV using a Sindbis virus-based heterologous gene expression vector, the pSinRep5. We designed two expression vectors (pSinRep5/JEV C-E and pSinRep5/JEV C-NS1), which encode the precise coding sequence of JEV C-E and JEV C-NS1 proteins, respectively. These cloned JEV structural protein genes were designed to express under the Sindbis virus subgenomic promoter. Upon the transfection and expression of the pSinRep5/JEV C-E or pSinRep5/JEV C-NS1 plasmid, the transfected cells expressed approximately 55 kDa JEV E prtiens. As designed, the JEV NS1 proteins were expressed only in the SinRep5/JEV C-NS1 RNAtransfected cells. In addition, we found in the pSinRep5/JEV C-NS1-transfected cells that the viral proteins were predominantly localized around the perinuclear membranes. On the other hand, cytoplasmic staining was mainly observed in the pSinRep5/JEV C-E RNA-transfected cells in the absence of NS1 protein. Thus, our system will provide a useful tool to dissect intracellular membrane localization signals located in the JEV structural proteins without handling the infectious JEV viral particles and to characterize viral morphogenesis of this pathogen.
Asian Continental Ancestry Group* ; Child ; Clinical Coding ; Clone Cells ; Cytoplasm ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Gene Expression ; Hand ; Humans ; Intracellular Membranes ; Membranes ; Morphogenesis ; Mortality ; Plasmids ; Sindbis Virus ; Transfection ; Viral Proteins ; Virion

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