BACKGROUND: Because of a lack of substances for platelet (PLT) metabolism and preservation, normal saline (NS) washed PLTs can only be stored for short lengths of time. However, the use of platelet additive solutions (PAS) could help solve this problem. In this study, the in vitro quality of NS washed platelets (wPLTs) stored in two types of PAS were compared with those of wPLTs stored in NS. METHODS: Five units of NS washed apheresis platelets were pooled aseptically and separated into five aliquots for storage in NS only as well as T-PAS+ (Terumo BCT, Lakewood, CO, USA) and CompoSol PS (Fenwal, Lake Zurich, IL, USA) with or without 15 mM glucose. The parameters of wPLTs quality were assessed up to 48 hrs after washing and the whole experiment was repeated 10 times independently. RESULTS: wPLTs in two kinds of PAS had better quality than wPLTs in NS, and wPLTs in T-PAS+ showed better quality than those in CompoSol PS. PAS-stored wPLTs with added glucose maintained stable CD62P and Annexin V expression during storage, but exhibited increased lactate accumulation. Evaluation of in vitro quality revealed that all wPLTs had a rating of 4 immediately after washing. However, only T-PAS+-stored wPLTs with glucose maintained a rating of 4 up to 48 hrs of post-washing. CONCLUSION: Using PAS storage for wPLTs may be beneficial compared to NS. The results presented herein suggest that T-PAS+ containing glucose has the potential to extend storage time by up to 48-hours.
Annexin A5 ; Blood Component Removal ; Blood Platelets* ; Blood Preservation ; Glucose ; In Vitro Techniques ; Lactic Acid ; Lakes ; Metabolism

Annexin A5 ; biosynthesis ; isolation & purification ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo investigate the expression of annexin V in the decidua tissues of preeclampsia patients and explore its clinical significance.

METHODSReal-time PCR, Western blotting and immunohistochemistry were employed to detect the mRNA and protein expressions of annexin V in the deciduas from 35 normal pregnant women at full term, 38 early onset severe preeclampsia patients and 33 late onset severe preeclampsia patients.

RESULTSAnnexin V was found on the cell membrane and in the cytoplasm of the decidual cells and stroma. Both the mRNA and protein of annexin V expressions in the decidua tissues were significantly different between normal pregnancy group and early or late onset severe preeclampsia group (P<0.05), being the highest in normal pregnancy group and the lowest in early onset severe preeclampsia group.

CONCLUSIONThe low expression of annexin V in the deciduas might participate in the hypercoagulability state in preeclampsia patients.

Adult ; Annexin A5 ; metabolism ; Case-Control Studies ; Decidua ; metabolism ; Female ; Humans ; Immunohistochemistry ; Pre-Eclampsia ; metabolism ; pathology ; Pregnancy ; RNA, Messenger ; genetics

OBJECTIVETo quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions.

METHODSThe cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0 micromol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V-FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably representlate stage of apoptosis, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, Annexin+ /PI- and Annexin+ /PI+ cells were sorted by fluoresence-activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electrophoresis.

RESULTSThe percentage of apoptotic cells was found to increase with the incubation time (r = 0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV) (4.2%). Meanwhile, the Annexin+ /PI- and Annexin+ /PI+ cells were identified as apoptotic and necrotic cells under EM, and DNA extracted from the Annexin+ /PI- cells was characterized by "ladder pattern".

CONCLUSIONSAnnexin-V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.

Annexin A5 ; analysis ; Apoptosis ; Burkitt Lymphoma ; pathology ; DNA Damage ; Humans ; Necrosis ; Phosphatidylserines ; metabolism ; Propidium ; analysis ; Tumor Cells, Cultured

The role of surfactant protein A (SP-A) in the recognition and clearance of apoptotic cells is well established, but to date, it is still not clear which surface molecules of apoptotic cells are involved in the process. Here we present evidence that phosphatidylserine (PS) is a relevant binding molecule for human SP-A. The binding is Ca(2+)-dependent and is not inhibited by mannose, suggesting that the sugar-binding site of the carbohydrate recognition domain (CRD) of SP-A is not involved. Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells, and this was consistent for Jurkat cells and neutrophils. Supporting these data, confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells. However, we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface, as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.
Annexin A5 ; metabolism ; Apoptosis ; Carboxy-Lyases ; metabolism ; Flow Cytometry ; Humans ; Jurkat Cells ; Microscopy, Confocal ; Neutrophils ; physiology ; Phosphatidylserines ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism

OBJECTIVETo study luteolin-induced non-small cell lung cancer cell line A549 apoptosis and the molecular mechanism for inhibiting its cycle arrest (G2 stage).

METHODMTT assay showed that luteolin had obvious inhibitory effect on A549 and indicated the half inhibition ratio (IC50). Cell cycle and apoptosis were detected by Hoechst 33258 nuclear staining assay, Annexin V-FITC/PI double staining and flow cytometry. Western blotting assay revealed changes in cycle and apoptosis-related proteins induced by luteolin. Possible molecular mechanism was suggested by Western blotting and immunocytochemistry.

RESULTLuteolin had an obvious growth inhibitory effect on A549 cells, with IC50 of 45.2 micromol x L(-1) at 48 h. Flow cytometry showed A549 cells mainly arrested in G2 stage after being treated by luteolin, with low expressions in cyclin A, p-CDC2 and p-Rb. Hoechst 33258 nuclear staining and Annexin V-FITC/PI double staining showed that the luteolin treatment group showed a significant apoptosis rate than the non-treatment group. Western blotting found luteolin can increase phosphorylation of JNK and decrease that of NF-kappaKB (p65). Immunocytochemistry results revealed luteolin can inhibit TNF-alpha-stimulated p65 from nuclear translocation as a transcription factor and thus promoting cell apoptosis.

CONCLUSIONLuteolin can obviously induce apoptosis of human non-small cell lung cancer cell A549 possibly by increasing phosphorylation of JNK to activate mitochondria apoptosis pathway, while inhibiting NF-kappaB from nuclear translocation as a transcription factor.

Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Humans ; Luteolin ; pharmacology ; NF-kappa B ; metabolism

OBJECTIVETo study the differentially expressed proteins in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells.

METHODSPrimary rat Leydig cells were cultured in vitro and treated with annexin 5 at the concentration of 1 nmol/L for 24 hours, and the cell proteins were extracted to be compared by two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots were selected to be analyzed by mass spectrometry.

RESULTSWe obtained electrophoresis profiles with high resolution and reproducibility, found 50 differentially expressed protein spots, and identified 36 by mass spectrometry, of which 23 were overexpressed and 13 underexpressed in the Leydig cells treated with annexin 5.

CONCLUSIONDifferentially expressed protein profiles were established in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells, and identified the key role of these proteins in testosterone secretion. Our findings might be helpful to illuminate the mechanism of annexin 5 regulating testosterone secretion in rat Leydig cells.

Animals ; Annexin A5 ; pharmacology ; Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Leydig Cells ; drug effects ; metabolism ; Male ; Mass Spectrometry ; Proteins ; metabolism ; Proteome ; analysis ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

OBJECTIVETo evaluate the effect of sensitized sera on engraftment of hematopoietic stem cells (HSCs) and its mechanism.

METHODSBone marrow cells (BMCs) from C57BL/6 mice were incubated with sensitized sera (group I) or normal sera (group II), and then washed and transplanted into irradiated BALB/c mice. The survival analysis and engraftment evaluation of the recipients were observed. The incubated BMCs were bound with goat anti mouse IgG for and labeled with Annexin V for apoptosis detection.

RESULTSSeven out of ten recipient mice in group I died of bone marrow failure at day 10 after transplantation, while all of those in group II were long-term survived. Engraftment assay showed recipients blood count and BMCs progressively decreased along with time passing in group I; in addition, the chimeric percentage of donor cells progressively decreased as well. The percentage of BMCs binding with goat anti mouse IgG in group I or group II were (90.3 +/- 5.1)% and (5.2 +/- 2.4)%, respectively, and the difference was statistically significant (P < 0.01). However, no significant difference was found in the apoptosis detection between the two groups (P > 0.05).

CONCLUSIONThe engraftment capacity of HSCs is significantly impaired by sensitized sera, the antibodies in sensitized sera may bind to antigens expressed on HSCs but do not induce apoptosis.

Animals ; Annexin A5 ; metabolism ; Apoptosis ; Bone Marrow Cells ; metabolism ; pathology ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Immune Sera ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVETo investigate the expression of human annexin-V (HA-V) in relation to HBV infection in different fetal tissues.

METHODSImmunohistochemistry was employed to detect the expression and distribution of HA-V in the liver, kidney, ovary, heart, fallopian tube, spleen, and thymus gland of human fetus.

RESULTSHA-V expression was detected in different tissues including the ovary, liver, intrahepatic bile duct, heart, kidney, lymphocytic cells in the thymus gland, epithelial cells of the fallopian, and cortical and medullary cells of the spleen. HA-V was distributed mainly in the cytoplasm of the cells. The liver tissues exhibited greater gray scale for HA-V expression than in the other tissues (P<0.05) and no significant difference was observed in the other tissues than the liver (P>0.05) in image analysis with Photoshop 7.0.

CONCLUSIONHA-V is an inherent protein in fetal tissues with possible relation to HBV infection of different tissues as a HBV receptor. Greater amount of HA-V in the liver may account for the vulnerability of the liver to HBV infection.

Annexin A5 ; analysis ; Fetus ; chemistry ; virology ; Hepatitis B ; metabolism ; virology ; Hepatitis B virus ; growth & development ; Humans ; Immunohistochemistry ; Liver ; chemistry ; virology ; Tissue Distribution

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons.
Acetylcholinesterase ; Adenoviridae ; AMP-Activated Protein Kinases ; Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; Cell Death ; Cell Survival ; Energy Metabolism ; Hippocampus ; Memory ; Neurons ; Neuroprotective Agents ; Rats ; Scopolamine Hydrobromide ; United Nations