The expression of oxidized low density lipoprotein and scavenger receptor that binds phospha-tidylserine and oxidized lipoprotein in patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the expression level of oxidized low density lipoprotein (ox-LDL) and its scavenger receptor scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SRPSOX) in patients with rheumatoid arthritis (RA),and to explore the relationship between ox-LDL and disease activity.Methods The serum ox-LDL in RA patients and healthy control group were detected by enzymelinked immunosorbent assay (ELISA),as well as the fluidox-LDL in RA,osteoarthritis (OA) and inflammatory arthritis (IA).The expression of SR-PSOX in mixed cells of RA and IApatients was detected by western blot.The expression of serum ox-LDL between RA groupand the control group was analyzed by t-test and non-parametric test.The correlation of serum ox-LDL expression levels in RA patients with C-reactive protein (CRP),erythrocyte sedimentation rate (ESR) and other inflammatory factors and disease activitywas analyzed by Pearson linear regression.Results The expression of ox-LDL in the serum of RA patients was significantly higher than that of normal control group [(3 076±131) mU/ml,(2 334±84) mU/ml,t=4.242,P＜0.01].The expression of ox-LDL in synovial fluid of RA patients was significantly higher than that of the OA group [(4 963±354) mU/ml],(3 956±347) mU/ml,t=2.372,P＜0.05).The expression of SR-PSOX in synovial fluid mixed cells of RA patients was higher than that of the IA group [(4.92±0.18) vs (0.24±0.04),t=33.53,P＜0.01].The expression of ox-LDL in serum of RA patients was negatively correlated with ESR,CRP and overall disease activity DAS28 (r=-9.42,P=0.009;r=-0.35,P=0.029 7;r=0.42,P=0.008 4).The expression of ox-LDL in the serum of RA patients with moderate disease activity was significantly higher than those patients with high disease activity [(3 302±138) mU/ml vs (2 464±228) mU/ml,t=3.335,P＜0.01],however,those with low disease activity and disease remission had higher serum ox-LDL expression but without statistical significant differences.After treated with anti-rheumatic drugs (DMARDs),serum ox-LDL of RA patients had a trend of slight increasing butwithout sign-ificant difference.The ox-LDL/LDL-C or ox-LDL/HDL-C was negatively not correlated with disease activity score in 28 (DAS28),ESR,CRP.Conclusion In RA patients,the expression of ox-LDL in the serum and synovial fluid is high and the SR-PSOX expressionin synovial fluid is also high.The serum ox-LDL levels are negatively correlated with ESR,CRP and DAS28,which are related to disease activity of RA.These findings suggest that the ox-LDL and the receptor SR-PSOX may play a role in RA pathogenesis,but needs further study.

Value of anti-phosphatidylserine/prothrombin antibody in diagnosis of seronegative antiphospholipid syndrome / 上海交通大学学报（医学版）

Objective • To investigate diagnostic value of anti-phosphatidylserine/prothrombin antibody (aPS/PT), IgA anti-cardiolipin antibody (aCL), and IgA anti-β2-glycoprotein antibody (aβ2-GPI) in seronegative antiphospholipid (SNAPS). Methods • Serum samples were collected from 86 patients with antiphospholipid (APS) (APS group), 48 patients with SNAPS (SNAPS group), 79 patients with systemic lupus erythematosus (SLE) (SLE group), and 85 healthy donors (healthy control group, HC group) for aPS/PT (IgG and IgM), aCL (IgA) and aβ2-GPI (IgA) detected by ELISA. The sensitivity and specificity of the four antibodies for the diagnosis of SNAPS were calculated, and the ROC curves were analyzed. The correlation between the four antibodies and the clinical manifestations of SNAPS was also analyzed.Results • A total of 25 (52.1%) SNAPS patients were positive in aPS/PT (IgG/IgM), including 29.2% patients positive in aPS/PT (IgG) and 35.4% positive in aPS/PT (IgM). There were 16.7% SNAPS patients positive in aβ2-GPI (IgA), but none was aCL (IgA) positive. The positive rates of aPS/PT (IgG and IgM) and aβ2-GPI (IgA) were statistically higher in SNAPS group than those in HC group (P=0.000). The area under curve (AUC) of aPS/PT (IgG) (AUC=0.753) for SNAPS diagnosis was the biggest among the four antibodies, and the second was aβ2-GPI (IgA) (AUC=0.725). A positive correlation was found in SNAPS group between presence of venous thrombosis and aPS/PT (IgG) (OR=5.54, 95% CI 1.67-17.33, P=0.003) or aβ2-GPI (IgA) (OR=3.43, 95% CI 0.86-11.53, P=0.041), and also found between pregnancy loss and aPS/PT (IgM) (OR=5.11, 95% CI 1.31-21.29, P=0.004). Conclusion • aPS/PT (IgG/IgM) and aβ2-GPI (IgA) can be used as potential complementary indicators for laboratory diagnosis of SNAPS, and aPS/PT (IgG/IgM) is also valuable for clinical evaluation of the risk of thrombosis and pregnancy loss in SNAPS patients.

"An apoptotic ""Eat Me"" signal: phosphatidylserine exposure" / 中国生化药物杂志

Phosphatidylserine (PS) is a phospholipid that is abundant in eukaryotic plasma membranes,has crucial biological functions.Under cell apoptosis, cells can not generate enough ATP for energy and the concentration of cytoplasmic Ca 2 +increases, resulting in PS eversion.Apoptosis and the clearance of apoptotic cells are essential processes in animal development and homeostasis.For apoptotic cells to be cleared, they must display aneat me signal, most likely PS exposure, which prompts phagocytes to engulf the cells.PS is exposed by the action of scramblase on the cell's surface in biological processes such as apoptosis and platelet activation.Once exposed to the cell surface, PS acts as an eat me signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets.The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers.Indeed, their identity as well as the mechanism of the PS exposure to the cell surface has only recently been revealed.We describe how PS is exposed in activated platelets and in apoptotic cells, and discuss the clearance of apoptotic cells.

Oviductal epithelial cells selected boar sperm according to their functional characteristics / 亚洲男科学杂志(英文版)

The interaction of oviductal epithelial cells (OECs) with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization (IVF), tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF: (i) W30 group: spermatozoa were incubated with oocytes in the absence of OEC; (ii) NB group: after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and (iii) B group: after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates (NB: 19.6%, B: 94.9%, and W30: 62.9%; P < 0.001) and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups (NB: 58.7%, B: 2.5%, and W30: 4.5%; P < 0.01). A similar trend was observed in phosphatidylserine translocation (NB: 93.7%, B: 5.7%, and W30: 44.2%; P < 0.01). These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

In vivo pharmacokinetic study on curcumin-loaded nanostructured lipid carriers in rat based on UPLC-MS/MS analysis / 中草药

Objective: To study the pharmacokinetics of phosphatidylserine (PS)-containing curcumin-loaded nanostructured lipid carriers (Cur-mNLC), curcumin-loaded nanostructured lipid carriers without PS (Cur-NLC), and curcumin solutions in SD rats. Methods: Blood samples were collected from the orbit of rats at different periods of time after they were ip injected with Cur-solution, Cur-NLC, and Cur-mNLC at 10 mg/kg (corresponding to Cur), then Cur content in plasma was determined by UPLC-MS/MS, and the pharmacokinetic parameters were calculated by DAS software. Results: The pharmacokinetic parameters of Cur-solution, Cur-NLC, and Cur-mNLC were respectively calculated as follows: Cmax (29.453±1.146), (20.045±0.818), (15.865±0.409) μg/L; MRT(0-∞) (18.196±1.456), (18.196±1.456), (89.252±12.049) h. The AUC(0-∞) of Cur-mNLC was 2.58 and 1.48 times larger than those of Cur-solution and Cur-NLC. Conclusion: The Cmax of Cur-NLC and Cur-mNLC are lower than that of Cur-solution, but the MRT(0-∞) of Cur-NLC and Cur-mNLC are longer than that of Cur-solution. Compared with Cur-NLC, Cur-mNLC has better sustained-release behaviors, which contributes to its superior bioavailability.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Phosphatidylserine exposure in red blood cell in sepsis patients / 国际生物医学工程杂志

Objective To investigate the morphologic change and phosphatidylserine (PS) exposure of erythrocytes in sepsis patients.Methods 30 healthy volunteers (control group)and 30 sepsis patients were enrolled in this study and were collected venous sampling.Monitoring included Wright's staining blood smear test,erythrocyte aggregation index and the ratio of PS exposure of erythrocytes.A flow-cytometric assay based on FITC-Annexin V was used to measure the PS exposure of erythrocytes.Results The morphological changes of red blood cells included acanthocyte,lachrymiform,rouleaux,spherocyte in sepsis patients,and the peripheral blood erythrocyte aggregation and aggregation index were significantly higher than that of the healthy control group (P＜0.05).The percentage of PS exposure of erythrocytes in sepsis patients were significantly higher than that of healthy volunteers (P＜0.001).Conclusion The PS exposure of erythrocytes were significantly higher in sepsis patients,and the morphology of red blood cells is obvious abnormal.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

Effect of parathyroid hormone on renal anemia in patients with chronic kidney disease / 中华肾脏病杂志

Objective To observe the mechanism of elevated parathyroid hormone(PFH) leading to erythrocytes life span shortened in the patients with chronic kidney disease(CKD).Methods Serum samples of 30 healthy people and 75 CKD patients were collected.Patients were divided into three groups according to their renal function.Intact parathyroid hormone(iPTH)was detected by immunochemiluminometry.The erythrocytes phosphatidylserine (PS) exposure and intracellular calcium concentration ([Ca2+]i)were measured by flow cytometry. Results (1)Levels of serum iPTH,[Ca2+]i and erythrocytes PS exposure increased gradually with the decline of renel function in stages 3 to 5 of CKD,the differences were significant with CKD 1 to 2 stages and healthy control group(all P＜0.05).(2)Pearson correlation analysis revealed,during CKD 3 to 5 stages,Hb was negatively correlated with iPTH and erythrocyte PS exposure respectively (r=-0.830 and-0.791,all P＜0.01);iPTH was positively correlated with[Ca2+]i and erythrocyte PS exposure (r=0.882 and 0.924,all P＜0.01),whereas negatively correlated with serum Ca respectively(r=-0.544,P＜0.01);erythrocyte PS exposure was positively correlated with[Ca2+]i(r=0.923,P＜0.01)and not correlated with serum Ca(r=-0.138,P=0.365).(3)The linear regression equation of[Ca2+]i(Y)for iPTH(X)was Y=3.327+0.213X(F=21.529,P＜0.05).The multiple linear regression equation of erythrocytes PS exposure(Y)for PTH (X1)and[Ca2+]i(X2)was Y=-0.303+0.283X2+0.139X1(F=6.59,P＜0.01). Conclusions By increasing intracellular calcium,iPTH can lead to an increase of the erythrocyte PS exposure.which will cause the occurrence of erythrocytes life span being shortened.As a result,the renal anemia will become more severe.

The measurement of affinity of site-specific labeled annexin V with ~(99m)Tc by calcium titration / 基础医学与临床

Objective To derermine the affinity of site-specific labeled annexin V with ~(99m)Tc to phosphatidylserine (PS)exposed erythrocytes.Methods The annexin V fused with a metal chelating binding site was obtained from Pichia Pastoris culture and methanol induction expression.The annexin V was purified from the culture supernatant crude product by uhrafihration.The annexin V was conjugated with ~(99m)Tc site-specifically through sodium glucoheptonic acid and SnCl_2.The radioactive annexin V was added to determine its affinity to PS exposed erythrocytes.Results The calcium concentration at which half of the protein is bound to cells(EC50)is changed with varying ratio of protein to cells.The affinity was determined at a low protein/cell ratio as 33.4.Conclusion The annexin V recombinantly expressed in Pichia Pastoris shows high affinity to PS exposed erythrocytes.

The Conjugation of Fluorescein Isothiocyanate to Annexin V and its Application / 医学研究杂志

Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.

Cloning of the Phosphatidylserine Synthase Gene and Its Secretive Expression in Bacillus subtilis / 中国生物工程杂志

The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal-(ExPASy P23830) was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in the lab, and the recombinant plasmid pBES-pss was transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 (pBES-pss) was grown on Bacillus subtilis common fermentation medium, which contained 30?g/ml kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/ml. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.

Study of Hydroxyurea Induced Caspase Activities in Chronic Myelogenous Leukemic Cell Line, K562 cells / 대한임상병리학회지

BACKGROUND: Chronic myelogenous leukemia is a chronic myeloproliferative disorder characterized by leukocytosis with myeloid elements at all stages of differentiation, t(9;22)(q34;q11) and bcr/abl rearrangement. We studied hydroxyurea induced apoptotic changes such as externalization of phosphatidylserine, caspase activities on human chronic myelogenous leukemic cell line, K562 cells. METHODS: K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and treated hydroxyurea. Viability was examined by MTT assay. Apoptosis were examined by annexin V stain, caspase (such as caspase-, caspase-, caspase-, caspase-, and caspase-) activities, and DNA fragmentation. RESULTS: The viability of K562 cells were markedly decreased in a dose dependent manner of hydroxyurea. Phosphatidylserine externalization was detected by annexin V stain after 3 hours in hydroxyurea treated K562 cells and the value of lactate dehydrogenase was not significantly changed in their culture media. The upstream effector of caspase- was slightly increased and had influenced on caspase-. And downstream acting caspase protease of caspase- was markedly increased in a time dependent manner at hydroxyurea treated K562 cells. In addition, however the activities of caspase- and caspase- were not increased. We also found DNA fragmentation at hydroxyurea treated K562 cells between 48 hours and 72 hours on agarose gel electrophoresis. CONCLUSIONS: Hydroxyurea induces apoptotic change in K562 cells via externalization of phosphatidylserine, activations of caspase-, caspase-, caspase- proteases, and DNA fragmentation.

In Vitro Analysis of Apoptotic Cell Death Using Proton Nuclear Magnetic Resonance Spectroscopy / 대한암학회지

PURPOSES: Cells undergoing apoptosis display profound morphologic and biochemical changes in the nucleus and cytoplasm, loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine (PS) at the surface of the cell, membrane blebbing, and decreased membrane microviscosity. Proton nuclear magnetic resonance spectroscopy ('H NMR spectroscopy) is able to detect the mobile fraction of lipids contained in the cell, and thus is sensitive to membrane fluidity modifications related to lipid composition changes. We have used 'H NMR spectroscopy in HL-60 cell line to detect and characterize the changes in plasma membrane lipid associated with apoptotic cell death. MATERIALS AND METHODS: We performed annexin-FITC and propidium iodide dual fluorescence flow cytometry, DNA gel electrophoresis, and obtained 200 MHz 'H NMR spectra of the HL-60 cell cultures before and at 6, 12, 18, 24, 36 and 48 hours after the addition of doxorubicin (100 ng/mL). RESULTS: The onset of apoptosis is accompanied by a greater than four fold increase in signal intensity ratio of the membrane lipid methylene (-CH2) resonance (at 1.2 ppm) to the methyl (-CH3) resonance (at 0.9 ppm). The quantitative relationship between apoptosis and the H NMR signal intensity was determined by fluorescein-annexin V flow cytometry, and showed that increases in the CH2/CH3 resonance signal intensity ratio paralleled the surface expression of PS as an early marker of apoptosis ( y =0.80, N 18 samples). The gradual decrease in the ratio of choline resonance (at 3.2 ppm) to CH3 signal intensity after 12 hours in the time course' experiment is directly proportional to the percentage of apoptotic cells ( y =0.96, N=18 samples). CONCLUSIONS: Monitoring of the CH2 and choline resonance signal intensity may therefore be useful in detecting apoptosis. Further studies using various stimuli to induce apoptotic cell death will be necessary to better determine the capabilities of 'H NMR spectroscopy for the detection and estimation of apoptosis in vitro.

Functional and pathological significance of phospholipid asymmetry in erythrocyte membranes.

The normal asymmetric distribution of phospholipids in the plasma membrane is perturbed in erythrocytes from patients with chronic myelogenous leukemia. Since experimentally-produced lipid-symmetric erythrocytes are more interactive with cells of the reticuloendothelial system than are their lipid-asymmetric counterparts, the biological recognition of chronic myelogenous leukemia erythrocytes by the reticuloendothelial system was examined. With one exception, all erythrocyte samples from patients with chronic/benign chronic myelogenous leukemia were more adherent to endothelial cells and more readily phagocytosed by macrophages in vitro than were normal erythrocytes. Thus, these naturally occurring pathological erythrocytes display the same dysfunctional intercellular interactions as the laboratory models.

Effect of phospholipase-D on rat kidney mitochondria.

Incubation of purified rat kidney mitochondrial fraction with phospholipase- D resulted in the accumulation of phosphatidic acid in the membrane due to the degradation of membrane-bound phosphatidylcholine, -serine and -ethanolamine Simultaneously with the hydrolysis of the phospholipids, cholesterol and protein were released from the mitochondrial membrane into the medium, and binding of Ca2+ by mitochondrial membranes increased. Phospholipase Dtreated mitochondrial fraction exhibited increased swelling in vitro in the early stages of incubation (15 min) after which the mitochondria were ruptured. Membrane- bound adenosine triphosphatase was partially inactivated and the enzyme activity was not significantly restored by incubation with sonicated dispersions of phosphatidylcholine, -serine and cholesterol. These results indicate that removal of choline, serine and ethanolamine from membrane-bound phospholipids disrupt phospholipid-cholesterol and phospholipid-protein association and affect functions of the membrane.