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

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

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

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

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

AIMTo analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate(%FR).

METHODSSemen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) microm/s), straight line velocity (VSL) (micro m/s), curvilinear velocity (VCL) (microm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mciromol/L of 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 degrees C in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test.

RESULTSSignificant differences were found between the high and the low groups in %MOT (91.1+/-8.5 vs 63.0+/-32.7, mean+/-SD), VAP (73.0+/-14.2 vs 52.1+/-12.5), VCL (127.0+/-28.1 vs 87.0+/-22.6), %HA (27.3+/-23.6 vs 7.2+/-9.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters.

CONCLUSIONWhen the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.

Embryo Transfer ; Female ; Fertility ; physiology ; Fertilization in Vitro ; Flow Cytometry ; Humans ; Intracellular Membranes ; physiology ; Male ; Membrane Potentials ; physiology ; Mitochondria ; physiology ; ultrastructure ; Semen ; physiology ; Sperm Motility ; Spermatozoa ; physiology

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

To elucidate the possibility whether an elevation of intracellular Ca2+ concentration ([Ca2+]i) in rabbit coronary artery myocytes during ischemic cardioplegic period may serve as one of the mechanisms of the "no-reflow' phenomenon or not, the changes in [Ca2+]i were measured under ischemic cardioplegia conditions using a fluorescent Ca2+ indicator, fura 2/AM. When single cells were perfused with cardioplegic or ischemic cardioplegic solutions, [Ca2+]i was significantly increased and the degree of [Ca2+] elevation was further augmented by the ischemic cardioplegic solution. Pretreatment of a sarcoplasmic reticulum emptying agent, 20 mM caffeine, had no effect on ischemic cardioplegia-induced [Ca2+]i changes, but application of a Ca2+ channel blocker, 5 x 10 (-1)M nifedipine, or an antagonist of Na+/Ca2+ exchange, 5 mM Ni2+, significantly inhibited the [Ca2+]i elevation, respectively. The magnitude of ischemic cardioplegia-induced [Ca2+]i elevation was dependent on the Ca2+ concentration of perfusate in the range of 0 and 25 mM. When Ni2+ was added to the reperfusion solution, recovery of ischemic cardioplegia-induced [Ca2+]i elevation was very rapid compared with the controls. It is concluded that ischemic cardioplegia-induced [Ca2+]i elevation may serve as one of the mechanisms of the "no-reflow' phenomenon in rabbit coronary artery smooth muscle cells. We propose that Na+/Ca2+ exchange may serve as a key function in ischemic cardioplegia-induced [Ca2+]i elevation.
Animal ; Arteries/metabolism ; Calcium/*metabolism ; Coronary Vessels/*metabolism ; Female ; *Heart Arrest, Induced ; Intracellular Membranes/*metabolism ; Male ; Muscle, Smooth, Vascular/*metabolism/pathology ; Myocardial Ischemia/*metabolism/pathology ; Osmolar Concentration ; Rabbits ; Support, Non-U.S. Gov't

Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca2+ and Na+ activities of the rat and guinea pig ventricular myocytes with two specific aims; whether there are any differences in intracellular Na+ activities between rat and guinea pig ventricular muscle cells, and if any, how the differences in intracellular Na+ activities are related to the effect of Na(+)-Ca2+ exchange on the action potential configuration and excitation-contraction coupling of the rat and guinea pig ventricular myocytes. Ryanodine (10(-7) M) diminished the slow repolarization phase of the rat ventricular action potential while the duration of the rapid repolarization phase increased. Ryanodine (10(-7) M) significantly increased the plateau of the action potential. At the steady state of 0.2 cps, intracellular Na+ activities (aiNa) of the rat and guinea pig ventricular myocytes were 8.7 +/- 5.2 mM (n = 16, 4 rats) and 10.0 +/- 4.1 mM (n = 25, 7 guinea pigs) respectively, but there were no statistically significant differences. The contractility of the rat ventricular muscle nearly disappeared due to ryanodine (10(-7) M) with little changes in aiNa. Monensin (10 mM) not only increased the resting tension but also remarkably increased aiNa from 2.0 mM to 20 mM. Ryanodine (10(-7) M) continuously decreased aiNa of the guinea pig ventricular muscle after the contraction ceased to decrease. Monensin increased the contractility as well as aiNa. These results suggest that the contractility of rat and guinea pig ventricular myocytes is determined by the change in the action of the Na(+)-Ca2+ exchange mechanism depending upon the plateau of action potential and the intracellular Na+ and Ca2+ activities. So ryanodine could decreases the contractility via its effect on Na(+)-Ca2+ exchange transport which could be one of possible mechanisms of negative inotropism by ryanodine.
Action Potentials/drug effects ; Animal ; Female ; Guinea Pigs ; Heart/*drug effects ; Heart Ventricle ; Intracellular Membranes/metabolism ; Male ; Myocardial Contraction/*drug effects ; Myocardium/cytology/*metabolism ; Rats ; Ryanodine/*pharmacology ; Sodium/*metabolism ; Support, Non-U.S. Gov't

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