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31.
Crowe JH Tablin F Wolkers WF Gousset K Tsvetkova NM Ricker J 《Chemistry and physics of lipids》2003,122(1-2):41-52
Human blood platelets are normally stored in blood banks for 3-5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved. 相似文献
32.
Houde C Roy S Leung N Nicholson DW Beauchemin N 《The Journal of biological chemistry》2003,278(19):16929-16935
The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms. 相似文献
33.
Phosphatidylinositol 3-kinase-dependent,MEK- independent proliferation in response to CaR activation 总被引:1,自引:0,他引:1
Bilderback TR Lee F Auersperg N Rodland KD 《American journal of physiology. Cell physiology》2002,283(1):C282-C288
Although ovarian surface epithelial(OSE) cells are responsible for the majority of ovarian tumors, we knowrelatively little about the pathway(s) that is responsible forregulating their proliferation. We found that phosphatidylinositol3-kinase (PI3K) is activated in OSE cells in response to elevatedextracellular calcium, and the PI3K inhibitors wortmannin and LY-294002inhibited extracellular signal-regulated kinase (ERK) activation by~75%, similar to effects of the mitogen-activated protein kinase/ERK kinase inhibitor PD-98059. However, in assays of proliferation, we found that PD-98059 inhibited proliferation by ~50%, whereas wortmannin inhibited >90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERKactivation in response to calcium. These results demonstrate that ERKactivation cannot account for the full proliferative effect of elevatedcalcium in OSE cells and suggest the presence of an ERK-independent,PI3K-dependent component in the proliferative response. 相似文献
34.
The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays. 相似文献
35.
Youngman KR Franco MA Kuklin NA Rott LS Butcher EC Greenberg HB 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(5):2173-2181
The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues. 相似文献
36.
Audinot V Fabry N Nicolas JP Beauverger P Newman-Tancredi A Millan MJ Try A Bornancin F Canet E Boutin JA 《Cellular signalling》2002,14(10):829-837
Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR. 相似文献
37.
Stefanov K Nechev J Lavchieva-Nacheva G Nikolova N Seizova K Kwartirnikov M Lavchiev V Popov S 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,131(3):543-550
Total lipid, fatty acid and sterol composition of larvae and adults of Musca domestica was investigated before and after feeding on sucrose syrup or on the same syrup containing 1% lead nitrate. The effects of sucrose and of lead ions were found to be different. In larvae sucrose diet inhibited the fatty acid elongation and stimulated the first stages of their unsaturation. A significant increase of phytosterol concentrations was obtained. These changes increased the cell membrane permeability. The addition of lead caused a decrease of the fatty acid unsaturation, which decreased the cell membrane permeability. In adults the sucrose diet had no effect on the lipid and sterol composition, while the addition of lead decreased the cholesterol concentration. The composition of lipids and sterols also depends on the diet of larvae before pupation. The data obtained suggested that changes in lipid and sterol composition, which control the permeability of the cell membrane, might be an adaptive response of the organism to the changes of the environment. 相似文献
38.
Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage. 相似文献
39.
40.
Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase 总被引:4,自引:0,他引:4
Conus NM Hannan KM Cristiano BE Hemmings BA Pearson RB 《The Journal of biological chemistry》2002,277(41):38021-38028
Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation. The paradigm for activation of Akt involves phosphatidylinositol 3-kinase-dependent membrane localization followed by activating phosphorylation of Thr-308 and Ser-473. Many of the activating signals for Akt involve the stimulation of receptor and non-receptor tyrosine kinases, and the most potent activator known is the tyrosine phosphatase inhibitor pervanadate, highlighting a possible role for tyrosine phosphorylation in the regulation of the enzyme. In this study we show that activation of Akt by pervanadate or serum is associated with tyrosine phosphorylation of Akt. In addition, in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt was tyrosine-phosphorylated in the basal state, and this phosphorylation was further enhanced by both pervanadate and insulin-like growth factor-1. We have used NH(2)-terminal sequencing and phosphate release analysis to directly identify Tyr-474 as the site of tyrosine phosphorylation. Substitution of Tyr-474 with phenylalanine abolished tyrosine phosphorylation of Akt and resulted in up to 55% inhibition of Akt activation, indicating phosphorylation at Tyr-474 is required for full activation of the kinase. Our data identifies a novel regulatory mechanism for this pleiotropic enzyme that may be applicable to the AGC family of protein kinases given the conserved nature of the COOH-terminal hydrophobic motif containing Tyr-474. 相似文献