OBJECTIVEGonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells, and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion. This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro.

METHODSThe encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a. The expression of the fusion protein HIS-annexin 5 was induced by isopropyl-beta-D-thiogalactoside (IPTG) under the control of the T7 promoter, and the products were purified by affinity chromatography. The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test. Semen samples from 15 donors were assigned to a control and an annexin 5 group, the latter treated with recombinant annexin 5 at the concentration of 10(-8) mol/L. Sperm motility and the percentage of grade a + b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure, and the sperm ascending experiment was done after 20 min treatment.

RESULTSThe product of the synthesized target gene was 947 bp in length, and the inserted sequence corresponded to the published encoding sequence of rat annexin 5. The plasmid pET28a-annexin 5 was transformed into E. coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36,000, a purity of 95% and a high anticoagulant activity. Compared with the control group, sperm motility and the percentage of grade a + b sperm were increased by 40% (P < 0.01) and 21% (P < 0.01), respectively, after 20 min treatment with annexin 5, but neither showed any significant improvement after 60 min. The sperm ascending altitude was remarkably elevated after annexin 5 treatment, with extremely significant difference from the control group (37.84 +/- 6.35 vs. 49.5 +/- 12.27, P < 0.01).

CONCLUSIONAn annexin 5 recombinant expression vector was successfully constructed. The protein annexin 5 can be efficiently expressed in E. coli and effectively improve human sperm motility in vitro.

Animals ; Annexin A5 ; genetics ; pharmacology ; Genetic Vectors ; Humans ; Male ; Plasmids ; Rats ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Sperm Motility

OBJECTIVETo investigate the role of Annexin 5 in protecting human sperm membrane and DNA integrity.

METHODSWe collected 53 semen samples based on the criteria of sperm density > 20 x 10(6)/ml and motility > 60%, and divided them into an experimental group (2.5 microl 10(-6) mol/L Annexin 5 added to 47.5 microl semen), a negative control group (2.5 microl 1 mol/L Tris-HCl [pH 8.0, 25 degrees C] added to 47.5 microl semen), and a blank control group (2.5 microl 0.01 mol/L PBS [pH 7.4] added to 47.5 microl semen). After 20 minutes of incubation, we evaluated the sperm membrane integrity using the hypoosmotic swelling test and, after another 60 minutes of treatment with H2O2 at 2.5 microl 10.02 mol/L, measured the sperm nuclear DNA integrity by acridine orange fluorescent staining.

RESULTSAfter 20 minutes of treatment with Annexin 5, the experimental group showed extremely significant difference in the percentage of hypoosmotic swelling sperm ([66.17 +/- 12.02] %) from the blank control ([58.13 +/- 13.08]%, P < 0.01) and the negative control group ([59.94 +/- 11.91]%, P < 0.01), but there was no significant difference between the latter two. Treatment with H2O2 remarkably increased DFI in the experimental group (6.39 +/- 1.07) as compared with the blank control (11.16 +/- 1.16) and the negative control group (10.86 +/- 1.05, P < 0.01), but no significant difference was observed between the latter two.

CONCLUSIONAnnexin 5 can increase the percentage of hypoosmotic swelling sperm in vitro and protect sperm membrane integrity, and it can also protect sperm DNA from H2O2 damage.

Annexin A5 ; pharmacology ; Cell Membrane ; drug effects ; DNA ; DNA Fragmentation ; Humans ; Male ; Sperm Count ; Sperm Motility ; Spermatozoa ; drug effects

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 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 establish a methotrexate (MTX)-resistant choriocarcinoma cell line and to determine its biologic characteristics.

METHODSMTX-resistant cell line (JAR/MTX) was derived from human choriocarcinoma cell line JAR by exposed to intermittently and progressively increasing concentration of MTX. Drug sensitivity was detected by MTT; P-gp GST-Pi and PCNA expressions were detected by immunohistochemistry. Cell apoptosis was detected by flow cytometry (FCM) with PI/Annexin V stain. Growth rates and human chorionic gonadotropin (HCG) production were also measured.

RESULTSJAR/MTX cell line was established with stable MTX-resistance (resistance index to MTX was 7.3) and cross-resistant to TAX and VCR. Growthrate of JAR/MTX was lower than that of parent cell line JAR. Expression level of PCNA in JAR/MTX was lower than that in JAR (3.09+/-0.42 compared with 3.72+/-0.35, P<0.05), while GST-pi expression was higher. No statistical difference of P-gp expression existed between two cell lines. JAR/MTX secreted more HCG than JAR every 10(5) cells secreted (95.7+/-5.4 compared with 41.3+/-2.8)mIU after 48 h(P<0.01). The flow cytometry showed that the spontaneous and MTX induced apoptosis in JAR/MTX was significantly lower than that in JAR P<0.05.

CONCLUSIONJAR/MTX cell line presented stable resistant to MTX and cross-resistant to TAX and VCR, which might sever as a model in study of drug resistance in choriocarcinoma.

Annexin A5 ; analysis ; Apoptosis ; Cell Adhesion ; Cell Division ; Cell Line, Tumor ; Choriocarcinoma ; drug therapy ; genetics ; pathology ; Drug Resistance, Neoplasm ; Humans ; Methotrexate ; pharmacology

OBJECTIVETo investigate the effects of saponins from Anemarrhena asphodeloides Bunge (SAaB) (Botanical Name: Anemarrhena Asphodeloidis Rhizoma) on the growth of vascular smooth muscle cells (VSMCs).

METHODSCell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA). Cell aldose reductase (AR) activity, as well as the effect of Epalrestat and interleukin-1beta were also explored.

RESULTSWST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB (P<0.01). Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs (P<0.01). Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-1beta. Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat (P<0.01).

CONCLUSIONSAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.

Anemarrhena ; chemistry ; Animals ; Annexin A5 ; metabolism ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Fluorescein ; chemistry ; Microscopy, Confocal ; Muscle, Smooth, Vascular ; cytology ; drug effects ; growth & development ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.
Time Factors ; Rats, Sprague-Dawley ; Rats ; Propidium ; Neuroprotective Agents/*pharmacology ; In Situ Nick-End Labeling ; Hypoxia-Ischemia, Brain/*drug therapy/metabolism/pathology ; Cycloheximide/*pharmacology ; Brain Infarction/pathology/prevention & control ; Apoptosis/drug effects ; Annexin A5/metabolism ; Animals, Newborn ; Animals

OBJECTIVETo investigate the effects of a new anticoagulant, annexin V derivative (AND) on anticoagulation and antithrombosis.

METHODSHigh and low doses of AND were given to rabbits (groups 1 and 2 respectively) by intravenous (iv) bolus injections followed by half the respective AND doses by iv infusion over 2 hours. Control groups were iv given heparin (group 3) and saline (group 4) of the same volume and procedure as that in group 1 and 2. Blood cell count, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen level were examined before and 15, 30 and 60 min after iv bolus and 2 hours after the end of iv infusion. A 3.0 mm x 15 mm balloon was put into femoral artery to induce endothelial denudation 15 min after IV bolus and the blood pressure of femoral artery was monitored until the pulse pressure recorded 0 mm Hg when the vessel was occluded completely by a thrombus. The femoral arteries were collected and the thrombi were stripped off for measuring their lengths, wet and dry weights.

RESULTSAnticoagulation parameters: APTT at 15 min after iv bolus in AND group was significantly longer than that in group 4 (P < 0.05) but shorter than that in group 3 (P < 0.05); APTT and TT in group 3 were significantly longer than those in groups 1, 2 and 4. Fibrinogen: 0.70 mg/kg AND may decrease fibrinogen. Antithrombosis values: the wet and dry weights in AND groups were significantly lighter than those in group 3 and 4 (P < 0.05). The dry weight in high-dose AND group was remarkably lighter than that in low-dose group (P = 0.029). The length of thrombus in low-dose AND group was remarkably shorter than that in group 4 (P = 0.013), but not for group 3 (P > 0.05). It was remarkably shorter in high-dose AND group than in both group 3 (P < 0.001) and 4 (P = 0.015). The time when pulse pressure equaled to 0 was longer in AND group than in group 4 (P < 0.05), but not in 3.

CONCLUSIONAND is an effective anticoagulant and antithrombosis agent, the highest anticoagulation effect occurs at 15 min after IV bolus. Its anticoagulation effect is not more potent than that of standard heparin, while antithrombosis capacity is more effective. AND in treating thrombosis clinically might be promising.

Animals ; Annexin A5 ; administration & dosage ; pharmacology ; Anticoagulants ; administration & dosage ; pharmacology ; Blood Coagulation ; drug effects ; Disease Models, Animal ; Fibrinogen ; analysis ; Humans ; Injections, Intravenous ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Rabbits ; Random Allocation ; Thrombin Time ; Thrombosis ; prevention & control

The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.
Animals ; Annexin A5 ; Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Liposomes ; chemistry ; Melanoma, Experimental ; Mice ; Microscopy, Confocal ; Paclitaxel ; pharmacology ; Particle Size ; Polyethylene Glycols ; chemistry

BACKGROUNDThe side effects of cyclosporine therapy include thromboembolic complications. However, the mechanisms underlying the hypercoagulable state induced by cyclosporine are not fully understood. Cyclosporine binds to red blood cells (RBCs) with a high affinity in circulation and alters the membranes of RBCs. Therefore, we propose that such alterations in RBCs membranes play a role in cyclosporine-induced coagulopathy and this disorder may be rectified by lactadherin, a phosphatidylserine binding protein.

METHODSRBCs from healthy adults were treated with various concentrations of cyclosporine. Procoagulant activity of the RBC membrane was measured by the single stage recalcification time and confirmed by detection of tenase and thrombin assembly through enzymatic assays. Inhibition assays of coagulation were carried out in the presence of lactadherin, annexin V or antitissue factor. Phosphatidylserine exposure was detected by flow cytometry and confocal microscopy through binding with fluorescein isothiocyanate (FITC)-labeled lactadherin as well as FITC annexin V.

RESULTSRBCs treated with cyclosporine demonstrated increased procoagulant activity. Cyclosporine treatment markedly shortened the clotting time of RBCs ((305 +/- 10) seconds vs (366 +/- 15) seconds) and increased the generation of intrinsic factor Xase ((7.68 +/- 0.99) nmol/L vs (2.86 +/- 0.11) nmol/L) and thrombin ((15.83 +/- 1.37) nmol/L vs (4.88 +/- 0.13) nmol/L). Flow cytometry and confocal microscopy indicated that cyclosporine treatment induced an increased expression of phosphatidylserine on the RBC membrane. Lactadherin was more sensitive in detecting phosphatidylserine exposure of the RBC membrane than annexin V. The modulating effect of procoagulant activity was concomitant with and dependent on phosphatidylserine exposure. Blocking of phosphatidylserine with lactadherin effectively inhibited over 90% of FXa generation and prothrombinase activity and prolonged coagulation time.

CONCLUSIONSProcoagulant properties of RBCs membranes resulting from phosphatidylserine exposure may play an important role in cyclosporine-induced thrombosis. Lactadherin can be used as a sensitive probe for phosphatidylserine detection. Its high affinity for phosphatidylserine may provide a new approach for the treatment of cyclosporine induced thrombogenic properties.

Adult ; Animals ; Annexin A5 ; chemistry ; Cattle ; Cell Membrane ; drug effects ; metabolism ; Cells, Cultured ; Cyclosporine ; pharmacology ; Erythrocytes ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Membrane Glycoproteins ; chemistry ; Microscopy, Confocal ; Milk Proteins ; chemistry ; Phosphatidylserines ; chemistry ; metabolism ; Thrombosis ; chemically induced ; metabolism