Mutations of G38R and D40G cause amyotrophic lateral sclerosis by reducing Annexin A11 protein stability / 中南大学学报(医学版)

To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS).
 Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot.
 Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein.
 Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.

Time-dependent proteomic and genomic alterations in Toll-like receptor-4-activated human chondrocytes: increased expression of lamin A/C and annexins

Activation of Toll-like receptor-4 (TLR-4) in articular chondrocytes increases the catabolic compartment and leads to matrix degradation during the development of osteoarthritis. In this study, we determined the proteomic and genomic alterations in human chondrocytes during lipopolysaccharide (LPS)-induced inflammation to elucidate the underlying mechanisms and consequences of TLR-4 activation. Human chondrocytes were cultured with LPS for 12, 24, and 36 h to induce TLR-4 activation. The TLR-4-induced inflammatory response was confirmed by real-time PCR analysis of increased interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) expression levels. In TLR-4-activated chondrocytes, proteomic changes were determined by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectroscopy analysis, and genomic changes were determined by microarray and gene ontology analyses. Proteomics analysis identified 26 proteins with significantly altered expression levels; these proteins were related to the cytoskeleton and oxidative stress responses. Gene ontology analysis indicated that LPS treatment altered specific functional pathways including ‘chemotaxis’, ‘hematopoietic organ development’, ‘positive regulation of cell proliferation’, and ‘regulation of cytokine biosynthetic process’. Nine of the 26 identified proteins displayed the same increased expression patterns in both proteomics and genomics analyses. Western blot analysis confirmed the LPS-induced increases in expression levels of lamin A/C and annexins 4/5/6. In conclusion, this study identified the time-dependent genomic, proteomic, and functional pathway alterations that occur in chondrocytes during LPS-induced TLR-4 activation. These results provide valuable new insights into the underlying mechanisms that control the development and progression of osteoarthritis.

Screening and identification of proteins related to gastric cancer metastasis with comparative proteomics / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen and identify the proteins related with tumor metastasis of gastric cancer in a nude mouse model bearing orthotopic transplanted tumor.</p><p><b>METHODS</b>Zinc finger protein 139 (ZNF139)-specific siRNA was synthesized and transfected into gastric cancer cell line SGC7901, which was then screened by G418. ZNF139-siRNA-transfected cells, negative plasmid-transfected cells and untreated SGC7901 cells were orthotopically transplanted separately on the stomach wall of BALB/c nude mice. The primary tumors and metastatic lymph nodes were harvested to separate the proteins by 2-D fluorescence difference gel electrophoresis (2-D DIGE); after gel digestion, the differential proteins were subjected to liquid chromatography-mass spectrometry (LC-MS) for identification and their functions were analyzed. Western blotting was performed to verify the identified proteins.</p><p><b>RESULTS</b>ZNF139 expression was effectively inhibited in siRNA-transfected SGC7901 cells. ZNF139-siRNA-transfected cells showed obviously suppressed tumor growth with a lowered lymph node metastasis rate in nude mice compared with untreated cells and the negative control cells (P<0.05). Proteomic study with 2-D DIGE showed that fascin and hnRNPA2/B1 were down-regulated while ANXA1 was up-regulated in the primary tumors, and ANXA5 was down-regulated in the metastatic lymph nodes in ZNF139-siRNA-transfected group. Western blotting confirmed the results of proteomic analysis.</p><p><b>CONCLUSION</b>ZNF139 gene may promote lymph node metastasis of gastric cancer by regulating fascin, hnRNPA2/B1, ANXA1, and ANXA5.</p>

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

Background : Although honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 - a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done. Results : We found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent. Conclusions : These observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.(AU)

Effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepatocellular carcinoma cell line HepG2 / 华中科技大学学报（医学）（英德文版）

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepato-cellular carcinoma cell line HepG2 were elucidated. The human ANXA10 gene was subcloned into the lentiviral vector, PGC-FU, to generate the lentiviral expression vector, PGC-FU-ANXA10. The corrected ANXA10 was confirmed by endoenzyme digestion, and sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells, and lentiviral particles were produced. The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting. HepG2 cells were infected, and divided into PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group. The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively. Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively. The recombinant PGC-FU-ANXA10 vector was successfully constructed, the ANXA10 protein was detected by using Western blotting, and virus titer was 2×10(8) TU/mL. The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%. At 72 h after infection, the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05); the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%, significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05), but there was no significant difference between PGC-Fu group and HepG2 cell group; the apoptosis rate in PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group was (51.92±1.41)%, (19.00±1.12)% and (3.59±0.89)% respectively. The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05). The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells. The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.

Growth advantage of CD34+cells in trisomy 8 high risk mids cases despite enhanced apoptotic signals

The essence of myelodysplastic syndrome [MDS] pathogenesis is damage of colony forming unit [CFU]with numerous reports implicating apoptosis. While low risk MDS showed enhanced intramedullary apoptosis, high risk MDS was associated with cellular proliferation giving the abnormal clone a growth advantage. Ninteen high risk MDS patients were studied for cellular apoptosis of bone marrow progenitors using tn-color flow cytometric quantification of CD34/Annexin/PU cells. Presence of cytogenetic abnormalities was detected using conventional analysis and FISH while bone marrow mononuclear cells' [BMMNC] survivin expression was evaluated using immunocytochemical examination of bone marrow cytospin preparations. The capacity for hematopoietic colony formation was performed using long term stem cell cultures. High risk MDS cases showed significantly higher percent of both apoptotic CD34/Annex1n*/PI cells and anti-apoptotic survivin cells compared to controls with significantly higher percent among trisomy 8 cases. Trisomy 8 cells showed a significant positive correlation with percent of apoptotic CD34/Annexin/PI cells and capacity for colony formation. The latter was significantly lower in MDS patients negative for trisomy 8 as compared to normal controls, while that of trisomy 8 cases was comparable to controls. Although trisomy 8 cells are in a pro-apoptotic state, they are checked by the enhanced expression of antiapoptotic signals which provide them with their proliferative advantage

Programmed cell death of B lymphocytes is induced by ligation of integrin-associated protein [CD47]

As part of the Immunoglobulin [Ig] superfamily, the transmembrane glycoprotein CD47 forms a signalling complex with the CD23 receptor alphavbeta3 integrin, which stimulates cytokine synthesis and controls inflammation by regulating leukocyte activation and the phagocytosis of aging apoptotic leukocytes. To investigate the effects of anti-CD47 antibodies on a range of cell types at varying stages of development. Using flow cytometric analysis, KMS11 and H929 human B lymphocyte multiple myeloma cell lines were used to study the expression of CD47 and alphavbeta3 integrin as a means to explore the factors relating to the induction and resistance to apoptosis. We found that both cell lines expressed high levels of CD47, with KMS11 cells expressing more than H929 cells. CD47 stimulated an increase in apoptosis in both KMS11 and H929 cells with the preponderance being late apoptotic, dying cells. Ligation of CD47 via the soluble phase was crucial for apoptosis to take place. These results provided an insight into the apoptotic mechanisms involved in the control of inflammation surrounding the activation and phagocytosis of leukocytes. In conclusion, further analysis is required to elucidate the complete signaling cascade responsible for this proliferative effect. Induction of apoptosis via CD47 stimulation appears to occur in the absence of CD47's signaling complex partner, alphavbeta3 whether or not apoptosis occurs, appears to be dependent upon the cell type and also the way CD47 is engaged

Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells.

According to the method used in our laboratory,our group synthesized (DIPP-Trp)2-Lys-OCH 3. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC 50 of 15.12 and 42.23 microM, respectively. (DIPP-Trp) 2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells;the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter,USA). Phosphatidylserine could signi?cantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells.The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining.It was concluded that (DIPP-Trp) 2-Lys-OCH3 not only induced cells to enter into apoptosis,but also affected the progress of the cell cycle.It may have arrested the K562 and HeLa cells in the G 2/M,S phases,respectively.The apoptotic pathway was pulsed at this point,resulting in the treated cells entering into programmed cell death.(DIPP- Trp)-Lys-OCH is a potential anticancer drug that intervenes in the signalling pathway.

Role of anti-annexin V antibodies in peripheral vascular ischemia of systemic sclerosis patients

To evaluate the role of a-AXVAbs IgG isotype in SSc and correlate it with peripheral microvascular and macrovascular affection. This study was conducted on 20 SSc female patients and 10 healthy controls. Clinical assessment was performed especially for peripheral vascular affection. ACL [IgG, IgM], Anti Scl-70 and a-AXV Abs were measured by ELISA and AC A by IIP. NFC and Doppler U/S were done. Diffuse SSc found in 11 patients and limited in 9 patients. A-AXV Abs were +ve in 19 patients, mean +/- SD 14.6 +/- 5.2, a highly significant increase compared to controls [t=5.3p<0.001]. There was no significant difference between diffuse and limited SSc [t-1.47 and p>0.05]. Patients were classified into 2 groups according to presence or absence of peripheral vascular affection: Group I, included 12 and Group II 8 patients. There was a highly significant difference between them regarding duration of RP [p<0.001], a significant difference regarding a-AXV Abs [p<0.05], but no such regarding ACL [IgG and IgM], anti-Scl-70 or anti-ACA. Within group I, there was a significant positive correlation between a-AXV Abs and disease duration, RP and ACL [IgG and, IgM] [p<0.05]. NFC showed scleroderma pattern: early 5%, active 75% and late 20%.Comparing a-AXV Abs in them, a highly significant difference in active and late [p<0.001]. Doppler US showed irregular thickening in carotid arteries, stenosis and calcification in ulnar and calcification and irregular thickening in femoral and popliteal arteries. A highly significant increase of a-AXV Abs in ulnar artery stenosis [p<0.001]. Measurement of a-AXV Abs in SSc can discriminate patients with vascular affection from those without it, with specificity 62.5%, and sensitivity 91.7%. Peripheral micro and macrovascular affection should be assessed

Phospholipid binding proteins antibodies: diagnostic importance and clinical associations in the antiphospholipid syndrome

Venous or arterial the thrombosis, abortion, and the presence of antiphospholipid antibodies [aPL] define the criteria for the antiphospholipid syndrome [APS]. The heterogeneous group of antibodies against phospholipids and plasma proteins may influence several coagulation pathways and lead to thrombophilia. Deep vein thrombosis and pulmonary embolism are the most common venous events. To establish whether antibodies directed against phospholipid-binding plasma proteins such as beta2-glycoprotein [beta2GPI], prothrombin [PT], and annexin V [Anx V] and antithrombin [anti-Thr] antibodies constitute a risk factor for thromboembolism in patients with primary antiphospholipid syndrome [pAPS] and systemic lupus erythematosus [SLE] and for miscarriage-in women with recurrent pregnancy loss [RPL], and whether their determination together with that aCL would help to increase the diagnostic sensitivity of aPL tests. The prevalence of various antibodies directed toward phospholipids [CL] and phospholipid-binding proteins [beta2GPI, PT, Anx V, and anti-Thr] was determined by immunoenzymatic method in 95 patients samples. Twelve patients with aCL-positive primary anti-phospholipid syndrome [pAPS]; 45 patients with SLE, 12 of whom had thrombotic complications [SLE/APS] and 33 of whom has no thrombosis; and 38 women with unexplained recurrent pregnancy loss comprised our study group. Fifty healthy subjects matched for age and sex were studied as the control group. Anti- beta2GPI, anti-PT, and IgG anti-Anx V anti-Thr antibodies were detected in 9[75%], 6 [50%],3[25%] and 3[25%], respectively, of the 12 aCL-positive pAPS patients; IgG and/or IgM aCL, anti- beta2GPI, anti-PT, IgG anti-Anx V and anti-Thr antibodies were detected in 17 [37%], 15 [33%], 20[44%], 6[13%] and 11[24%], of the 45 SLE patients respectively. The highest risk for thrombosis in SLE patients was associated with the presence of IgG anti-PT antibody [odds ratio [OR] 13.4, P<.001, vs. 7.7 aCL, 2.8 anti- beta2GPI, 0.6 anti-Anx V, and 8.7 anti-Thr]. Anti-Pt has slightly lower specificity than aCL however the occurrence of both antibodies brought the specificity 94%. In patients with recurrent pregnancy loss [RPL] the prevalence of IgG and/or IgM aCL, anti- beta2GPI, anti-PT, IgG anti-Anx V and anti -Thr antibodies was 6% [2], 12% [9], 6% [5], 16% [6], 18% [7], and 5% [2] respectively. Anti Anx V was the antibody significantly associated with miscarriage. The result of this study indicates that: It is useful to measure anti-PT antibodies in addition to the more widely used aCL and anti- beta2GPI antibodies in the prognostic evaluation of SLE patients for the risk of thrombosis. The results also confirm that anti-Anx V antibodies may play an important role in recurrent pregnancy loss. The determination of anti-thrombin antibodies may contribute to better diagnosis of APS

On-pump versus off-pump CABG: Study of platelet activation

Coronary revascularization on a beating heart avoids many of the side effects of cardiopulmonary bypass. Off-pump coronary bypass surgery offers an excellent chance to examine the impact of the surgery and the relatively high dose of heparin on platelet functions without using cardiopulmonary bypass. Sixty patients with coronary artery disease were prospectively randomized to [1] on-pump CABG with conventional cardiopulmonary bypass and worm cardioplegic arrest and [2] off-pump CABG on the beating heart. Platelet studies including bleeding time, platelet count, mean platelet volume, platelet aggregation using ADP and surface expression of P-selectin and Annexin-V using flow cytometry as markers of platelet activation. Platelet studies revealed definite platelet dysfunction in the on-pump group, however there was still statistically significant elevation of markers of platelet activation in the off-pump group. Platelet activation occurs in coronary bypass surgery even when CPB is no: used. This may correlate with previous reports of thrombotic complications of OPCAB surgery

Verificação da potência de extratos alergênicos comerciais de Dermatophagoides pteronyssinus para imunoterapia / Evaluation of the potency of commercially available Dermatophagoides pteronyssinus allergenic extracts for immunotherapy

Objetivo: Verificar a potência in vivo e in vitro de oito extratos alergênicos comerciais de Dermatophagoides pteronyssinus (Dp) para imunoterapia. Métodos: Foram adquiridos, sem o conhecimento dos fabricantes, os extratos mais concentrados utilizados em imunoterapia. Os alérgenos Der pie Der p 2 foram quantificados por método imunoenzimático, em oito extratos alergênicos de diferentes laboratórios. Os extratos foram avaliados por testes cutâneos de leitura imediata pela técnica de puntura (TCA) realizados com puntor descartável (Alko). Pacientes alérgicos a Dp (n=21O) e indivíduos não alérgicos (n=31) foram submetidos a TCA em seis serviços de alergia. A intensidade das reações foi aferida pelo diâmetro médio das pápulas, em leitura após 15 minutos. Resultados: Os níveis de alérgenos Der piou Der p 2 foram indetectáveis ou muito baixos (0,02 a 0,58 Ilg/mL) na maioria dos extratos, exceto no extrato I (68,3 e 31,7 Ilg/mL, respectivamente). Em pacientes alérgicos, a mediana dos diâmetros das pápulas obtidas em testes cutâneos foi < 3mm para 7/8 extratos analisados, enquanto que a mediana do diâmetro das pápulas obtidas com histamina foi de 6mm, e com o extrato I, de 7mm. A positividade dos testes (pápula > 3 mm) com os sete extratos variou de 2 a 36%. O extrato I teve positividade de 97,2%. Conclusão: A maioria dos extratos alergênicos testados in vitro e in vivo apresentou níveis de alérgenos insuficientes para alcançar as doses efetivas preconizadas internacionalmente para imunoterapia

Alterações da imunidade celular associada à patogênese da imunodeficiência Comun variável / Changes in cellular immunity asssociated with the pathogenisis of common variable immunodeficiency

Objetivo: Avaliação da morte celular por ativação em linfócitos T de pacientes com imunodeficiência comum variável (CVID) e de outros parâmetros da resposta imune. Métodos: Células mononucleares obtidas a partir de sangue periférico (PBMC) de 32 pacientes com CVID e 32 indivíduos normais foram utilizadas para o estudo da expressão de CD40L, linfoproliferação e apoptose, Para a análise de marcadores de ativação (CD25 e CD69) e de interação entre células T e B (CD70) PBMC foram estimuladas por diferentes tempos (24, 48, 72 e 96 horas), Os sobrenadantes de cultura foram utilizados para quantificação de citocinas (IL-2, IL-4, IL-5 e IFN-y) por ELISA. Todos os testes laboratoriais foram aplicados no grupo controle de voluntários sadios. Os resultados foram analisados utilizando testes de diferença de proporções, ANOV A, Kruskal-Wallis e a prova de Mann-Whitney. Resultados: O grupo de pacientes com CVID demonstrou aumento percentual de linfócitos CD3/CD4 que sofreram apoptose em relação ao grupo controle (p< (Au) pacientes. destes anticorpos por mediada e celular resposta da prejuízo conseqüente com Th2, citocinas de diminuída síntese além CD70, CD69 CD25, CD40L, expressão circulantes, B T células nas decréscimo pelo responsável seja fenômeno este que sugere ativadas morte aumento O Conclusões: normais. indivíduos grupo o comparados quando CVID pacientes.

cytotoxicity pathway of natural killer cells in cord blood compared to peripheral blood

The cytotoxic activity of natural killer cells is usually tested by radioactive assay [51Cr release assay], which detects the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, the assessment of cell damage by flow cytometry aims to provide a more exact characterization of the death pathway via detection of the percentage of apoptosis and necrotic cells. Annexin V-FITC [Axv -FITC] can be used to label cells in the early apoptopic state, while propidium iodide [PI] indicates late apoptosis or necrosis. The NK cytotoxicity of cord blood [CB] and peripheral blood [PB] was determined after 4 hours of incubation in the absence of cytokines. After 4 hours in vitro incubation, co-staining with Annexin V-FITC [Axv-FITC] and propidium iodide [PI] permitted discrimination between viable, early apoptotic and necrotic cells. As we would expect, the cytotoxicity pathway in PB mononuclear cells [MNCs] consists of both apoptosis and necrosis pathways but in CB MNCs it almost consists of early apoptosis; and necrosis is negligible. With escalating E: T [effector: target] ratio changes in the percentage of apoptotic cells in PB samples were significantly higher than CB samples. The mechanism [s] of the low cytotoxicity of resting cord NK cells is not well understood. Complementary research in this field is recognized to elucidate the phenotypical and functional properties of CB cells and how they relate to maturational stages. CB studies are important for transplantation research and may provide insight to the suppressive mechanism by which the host-recipient could evade GVHD and rejection

study of neutrophil apoptosis in systemic lupus erythematosis patients

The aim of this study was to find out the percentage of neutrophils apoptotic cells in the peripheral blood in patients with SLE and to determine its relation to disease activity. Twenty patients with SLE [Group I], and 10 disease control patients with rheumatoid arthritis [Group II] were studied. 10 normal control subjects with matched age and sex were also included. Serum antibodies to ds-DNA were measured by a commerical assay. The percentage of apoptotic peripheral blood neutrophils was determined by morphology and flow cytometric analysis of annexin v/propidium iodide labelled cells. There was a significant increase of apoptotic neutrophils assessed by morphology and annexin v in SLE patients compared with rheumatoid arthritis patients and normal controls. There was a positive correlation between serum antibodies to ds-DNA and the precentage of apoptotic neutrophils assessed by annexin V. Also, there were positive correlation between annexin v positive neutrophils and SLE activity measures SLAM. these results suggest that, increased percentage of apoptotic neutrophils in the peripheral blood could be a potential source of lupus specific autoantigens [anti-ds-DNA], and failure of its clearance could trigger or exacerbate SLE activity

The effects of differentiation and glucocorticoid treatment on the expression of lipocortin 1 in U937 cells / 알레르기

Lipocortin represents a family of similar Ca++ depentent phospholipid-binding proteins capable of blocking the activity of phospholipase A2 (PLA2) in vitro. Generally, these proteins are believed to inhibit the release of arachidonic acid from photopholipids and the formation of lipid mediators such as prostaglandin, leukotriene, and platelet activating factor. Lipocortin 1, initially identified as a glucocorticoid- responsive protein in macrophages and neutrophils has been implicated in transmembrane signal transduction during growth factor-mediated cell proliferation and transformation. To define the synthesis and its regulation, we investigated the expression of lipocortin 1 in both the mRNA and protein level in U937 cell line in the presence of several differentiation factors. The results were as follows. 1. The expression of lipocortin 1 and its mRNA was increased during TPA-induced differentiation of U937 cells to maximum of 2-fold and 5-fold respectively. Both the protein and mRNA levels decreased after 48 hours. 2. With the treatment with IFN-gamma, the expression of CD16 was increased. However, the protein and mRNA levels of lipocortin 1 were, not changed significantly. 3. Neither the dexamethasone or hydrocortisone have any effects on the expression of lipocortin 1 in both TPA-differentiated and undifferentiated U937 cells. The results from this study would give a light on defining the functional role of lipocortin 1 in macro-moncycle cell lineage and possibly some informative clues for the pathogenic mechanisms of the inflammatory diseases.

Lipocortin 1 mediates the suppressive effects of dexamethasone on CoNinduced proliferative response and nitric oxide production in rat splenic leukocytes

Lipocortin 1 has been proposed as a putative mediator of anti-inflammatory actions of glucocorticoids. We investigated the role of lipocortin 1 in the effect of dexamethasone using rat splenic leukocytes. Concanavalin A(ConA; 1-microgram/ml) increased the leukocyte proliferation and nitric oxide(NO) generation, which were measured as (3H)-thymidine uptake by the cells and nitrite accumulation in the culture media, respectively. Dexamethasone suppressed CoNinduced cell proliferation, in a concentration-dependent manner with EC-50 around 50nM. The addition of anti-lipocortin 1 (Anti-LC1) reversed dexamethasone effects: 0.24, 1.2, 6 microgram/ml of Anti-LC1 reversed dexamethasone(50nM)-induced suppression of thymidine uptake by 9+/-3%, 16+/-3%, 36+/-5%, respectively; 0.24, 1.2, and 6-microgram/ml of Anti-LC1 reversed dexamethasone-induced decrease of nitrite concentration by 49 +/- 16%, 61 +/- 20%, 77 +/- 19 %, respectively. The present data indicate that lipocortin 1 mediates, at least in part, glucocorticoids-induced suppression of leukocyte proliferation and blockade of NO generation.