Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.

AIM: To study the detail mechanism of interaction between PKC and GRK(2) and the effect of GRK(2) on activity of PKC. METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK(2) in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK(2) PH domain, GST-GRK(2) PH domain fusion protein and BTK PH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK(2) to PKC was also detected by western blot and Co-immunoprecipitation. RESULTS: The binding of GRK(2) PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain. CONCLUSION: GRK(2) can bind with PKC and down-regulated PKC activity.

[Effect of astrocyte-conditioned medium on tert-butyl hydroperoxide-induced apoptosis of PC12 cells].

OBJECTIVE: To investigate the effect of astrocyte-conditioned medium (ACM) on PC12 cell apoptosis induced by reactive oxygen species tert-butyl hydroperoxide (tbOOH). METHODS: PC12 cells attacked by tbOOH were cultured with Sprague Dawley rat cerebral cortex ACM, and the cell apoptosis was observed by fluorescent microscopy and transmission electron microscopy. The cell apoptosis rate was evaluated by flow cytometry and malondialdehyde (MDA) concentration measured using thiobarbituric acid (TBA)-reacting substances (TRABS). RESULT: TbOOH induced PC12 cell apoptosis, and ACM significantly decreased the apoptosis rate and MDA concentration in tbOOH-treated cells. CONCLUSION: ACM may increase the anti-oxidation capacity of PC12 cells and inhibit cell apoptosis induced by tbOOH.

[Effect of total Panax notoginseng saponins on the activity of human endothelial nitric oxide synthase gene promoter].

OBJECTIVE: To study the mechanism of total Panax notoginseng saponin (tPNS) in regulating the transcription activity of human endothelial nitric oxide synthase (heNOS) gene promoter. METHODS: With gene recombination technique, we subcloned the heNOS gene promoter sequence (from -1 to -1 600 bp) into the BglII/HindIII sites of the firefly luciferase reporter gene vector, pGL2-Basic (Promega), to yield the recombinant plasmid designated as peNOS-Luc. With lipofectamine- mediated co-transfection technique, peNOS-Luc, pGL2-Basic and pCMV-beta were cotransfected into NIH3T3 cells, which were treated with lipopolysaccharide (LPS), tPNS and transforming growth factor beta1 (TGFbeta1) respectively. The relative activities (Luc/beta-gal) were subsequently determined in the cell lysates to evaluate the effects of these 3 factors on the activity of heNOS gene promoter. RESULTS: Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmid, peNOS-Luc, was constructed correctly, which could be effectively expressed in NIH3T3 cells. Upon LPS stimulation, the luciferase activity was obviously decreased, contrary to the results of tPNS and TGFbeta1 treatment, and between the latter two agents, TGFbeta1 produced higher transcription activity. CONCLUSIONS: A firefly luciferase reporter gene vector containing heNOS gene promoter sequence has been constructed correctly. tPNS can up-regulate the activity of heNOS gene promoter in NIH3T3 cells.

Construction of signal peptide-FRET fusion expression vectors and their expressions in NIH3T3 cells.

OBJECTIVE: To construct the fluorescence protein vector pairs with toll-like receptor 4 (TLR4) signal peptide, namely pECFP-C1-SP and pEYFP-C1-SP, therefore to make the application of fluorescence resonance energy transfer (FRET) possible in the study of the membrane protein interaction during lipopolysaccharide (LPS) recognition. METHODS: pECFP-C1-SP and pEYFP-C1-SP were constructed by means of site-directed mutagenesis, and the constructed plasmids were transiently transfected into NIH3T3 cells via lipofectamin to observe their intracellular expressions under a fluorescence microscope. RESULTS: DNA sequence analysis attested the validity of the constructed fluorescence vectors with signal peptide for FRET, and the expression of the vectors was located principally on the cell membrane as observed under fluorescence microscope. CONCLUSION: The constructed vectors TLR4 signal peptide are valid and capable of expressing on the cell membrane, therefore they can be effectively used in the study of the interaction between the membrane proteins.

[Effect of Cobaltous chloride on the transcription activity of modified human eNOS promoter].

AIM: To construct modified promoter of human endothelial nitric-oxide synthase(eNOS) and investigate transcriptional activity of the eNOS promoter via a hypoxia model of human umbilical vein endothelial cells-12(HUVEC-12) stimulated by Cobaltous chloride in vitro. METHODS: The pGL2-eNOS-repSP3 vector was constructed by the SP1-replaced SP3 element with using DNA site-directed mutation of PCR. The cells were transfected with pGL2-eNOS-repSP3 vector and normal pGL2-eNOS-p vector, repsectively. Compared with another modified eNOS promoter(pGL2-eNOS-insSP1) the transcriptional activity of pGL2-eNOS-repSP3 vector was determined using a double luciferase reporter gene system by the stimulation of different concentrations of Cobaltous Chloride. RESULTS: The pGL2-eNOS-repSP3 vector was constructed successfully. After the stimulation of Cobaltous Chloride, the transcriptional activities of both pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1 increased as the concentration of Cobaltous Chloride increased. But there was not significantly statistical difference between them. CONCLUSION: There is no significant difference on transcriptional activities between the two modified vectors, pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1.

[Inhibitor of tyrosine phosphorylation AG490 inhibits the proliferation of Jurkat T cells].

AIM: To study effect of AG490 on the activation, proliferation, cell cycle, apoptosis and the expression of protein ICBP90 of Jurkat T cells. To explore the possibility of inhibiting and the initial growth mechanism of Jurkat T cells by blocking JAK signaling pathway. METHODS: The expression level of CD69 and CD25 on the surface of Jurkat T cells was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique. The proliferation of Jurkat T cells was analyzed by MTT method. The cycle of Jurkat T cell was analyzed by PI staining and Flow Cytometry. The apoptosis of Jurkat T cells was analyzed by Annexin V-FITC and PI staining. The expression of ICBP90 that correlated with the cell proliferation was checked by Western blot. RESULTS: The results came out that the proliferation capacity of Jurkat T cells was decreased with the increasing of AG490 ranging from 1 mmol/L to 30 mmol/L in a dose-dependent manner with the highest inhibitory rate of 27.37%. Cell-cycle analysis revealed that AG490 arrest it in G0/G1 phase and inhibited its entrance into S and G2/M phase, and reduced the expression of protein ICBP90, especially in the 24 h treatment. But the apoptosis of Jurkat T cells was not promoted by AG490. CONCLUSION: AG490 significantly inhibited the growth of Jurkat T cells, and its inhibition may reduce the expression of protein ICBP90, which was related to the cell cycle arrest rather than apoptosis.

[Effect of Jagged-1 on the cytokine expression of murine myeloid-derived dendritic cells].

AIM: To investigate the in vitro effect of soluble Jagged-1/Fc on the cytokine expression of murine myeloid-derived dendritic cells induced by both rmGM-CSF and rmIL-4. METHODS: The DC culture model under rmGM-CSF plus rmIL-4 conditions was established. The influences of Jagged-1/Fc on the morphology of DC was checked, and the cytokine expression were analyzed by Luminex system and ELISA. The activation capacity of Jagged-1/Fc-induced DC for allogeneic lymphocytes was detected by MTT assay. RESULTS: Compared with LPS or Zymosan A, the amount of IL-4 expression of Jagged-1/Fc-induced DC increased significantly and the amount of TNF-alpha expression decreased markedly but gamma-secretase inhibitor DAPT reversed the inhibition of Jagged-1/Fc on TNF-alpha expression from DC and returned it to the control level. Jagged-1/Fc altered the amount of IL-10, IL-6, IL-2, IL-12 and IFN-gamma expression unconspicuously and had no effect on TGF-beta expression. The mixed lymphocyte reaction(MLR) revealed that Jagged-1/Fc-induced DC had the weakest ability to activate allogeneic lymphocytes and LPS-induced DC showed the strongest activation ability. CONCLUSION: The results indicate that Jagged-1/Fc-induced DC incline to induce immune tolerance and to prime naïve T cells to the Th2 cells.

[An infrared imaging system for detecting electrophoretic mobility shift of DNA-protein complexes].

OBJECTIVE: To establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides. METHODS: GILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System. RESULTS: One lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load. CONCLUSION: This infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.

[Effect of a soluble Jagged 1/Fc chimera protein on the activation, proliferation and cell cycle of lymphocytes in mice].

AIM: To investigate effect of a soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) on activation, proliferation and cell cycles of lymphocytes in BALB/c mice. METHODS: A model to evaluate the lymphocyte proliferation stimulated with a polyclonal activator, concanavalin A (ConA), was established by a carboxy-fluorescein diacetate-succinimidyl ester (CFDA-SE)-labeling technique. Under an effective dose of 500 mug/L of Jagged 1/Fc, the effect of it on the lymphocyte proliferation was analyzed by flow cytometry. A propidium iodide-labeling technique was applied to estimate the influence of Jagged 1/Fc on the lymphocyte cell-cycle stimulated by phorbol 12,13-dibutyrate (PDB) plus Ionomycin (Ion). Expression levels of CD69 and CD25 molecules on surface of CD3(+) lymphocytes activated with ConA in the presence of Jagged 1/Fc were observed by a fluorescein-conjugated monoclonal antibody-labeling technique. RESULTS: Jagged 1/Fc had no effect on the expression levels of CD69 and CD25 of the CD3(+) lymphocytes stimulated with or without ConA, and no effect on the proliferation index of the lymphocytes stimulated by ConA or PDB plus Ion. It led to the increase of sub-G0 phase cell proportion and the decrease of S phase cell proportion of the lymphocytes, but did not influence the changes of the lymphocyte cell-cycle induced by PDB plus Ion. CONCLUSION: The results suggest that Jagged 1/Fc has no obvious effect on the activation and proliferation of lymphocytes in mice, but may promote the apoptosis of them, cause the G0/G1 phase arrest and block the S phase entry.

[Effect of anisomycin on allogeneic skin transplantation in mice].

AIM: To investigate the effect of anisomycin on the reactivity of lymphocytes and the rejection in the skin transplantation in mice. METHODS: The reactivity of T cells in unilateral or bilateral mixed lymphocyte reaction was detected by MTT assay. The skins from C57BL/6 mice were transplanted to BALB/c mice and the survival period of the grafts was assessed. The recipients were divided into three groups (physiological saline group, cyclosporin A group and anisomycin group). They were injected with physiological saline (0.1 mL), cyclosporin A (20 mg/kg) and anisomycin (12.5 mg/kg) separately once a day for 3 days before the operation and 7 days after the operation. RESULTS: With the optimal dose of 10.0 microg/L, anisomycin inhibited the reactivity of lymphocyte cells in both unilateral and bilateral mixed lymphocyte reactions. The survival period of the grafts from anisomycin group was longer than that from cyclosporin A group and physiological saline group. CONCLUSION: Anisomycin can inhibit the reaction of lymphocytes and prolong the survival period of the grafts in skin transplantation effectively. It could be used as a new way for transplantation rejection treatment.

[Effect of Jagged1 on the morphology of DC differentiation observed by atomic force microscopy].

AIM: To explore the effect of Jagged1 on the morphology of dendritic cell(DC) differentiation. METHODS: Mouse bone marrow cells were cultured in the presence of rmIL-4 and rmGM-CSF. Atomic force microscopy (AFM) was used to observe the ultrastructure of these DC. RESULTS: Similar to the control, the cells in Jagged1 group displayed irregular shape with spiny or sheet-like dendrites and the cellular surface was rough. In contrast, the cells in DAPT group exhibited little dendritic processes and most of them were of round shape and with smooth surface. Furthermore, the height of the cells in Jagged1 group was higher than that in control and DAPT group, while the diameter of the cells in both was roughly equivalent. CONCLUSION: Jagged1 has no significant effect on the morphology of DC, but significant changes in the morphology of DC in DAPT group have occurred.

[Effect of extracellular-signal regulated kinase 1/2 specific repressor PD98059 on murine lymphocyte proliferation].

AIM: To investigate the effect of PD98059, a specific repressor of extracellular signal regulated kinase 1/2 (ERK1/2), on the proliferation of the murine lymphocytes. METHODS: Lymphocytes were isolated from lymphoid nodes of BALB/c mouse. After stained with carboxy fluorescein diacetate, succinimidyl ester(CFDA-SE), the cells were stimulated with polyclonal activators, concanavalin(ConA) and phorbol 12,13-dibutyrate(PDB) plus ionomycin(Ion). The proliferation and cell-cycle of lymphocytes were analyzed using flow cytometry(FCM). RESULTS: CFDA-SE staining showed that 5, 10, 20, 30 and 40 micromol/L PD98059 could significantly inhibit the proliferation of murine lymphocytes induced by ConA. The inhibitory effect was dose-dependent(r=0.985, P<0.01). The optimal concentration of 30 micromol/L PD98059 was selected and its inhibitory effect was significantly decreased with prolongation of time (P<0.01); but the inhibitory rate increased. Analysis of the cell cycle induced by ConA revealed that the cells was blocked into S and G2/M phases as the concentration of PD98059 increased. However, the cell number in sub-G0/G1 peak (apoptotic peak) had no significant change under the concentration range used. PD98059 had the similar effect on the cells treated with PDB plus Ion, but S and G2/M phases were more distinct. CONCLUSION: PD98059 can inhibit the proliferation of the murine lymphocytes and blocked them into S and G2/M phases, indicating the activation of ERK1/2 signaling pathway plays an important role in the proliferation of lymphocytes.

[Real-time quantitative PCR for evaluating murine thymic function].

OBJECTIVE: To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function. METHODS: The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR. RESULTS: The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains. CONCLUSIONS: Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.

[The changes of the thymocyte mitochondria mediated by nitric oxide in thymocyte apoptosis].

AIM: To study the changes of mitochondrial potential (delta psi m) and cardiolipin (CL) content during thymocyte apoptosis mediated by nitric oxide (NO). METHODS: SNAP was used as NO's donor to induce thymocyte apoptosis in mice and dexamethasone (DEX) was used as positive control drug. Three experiment groups were set, which were blank control group, SNAP group and DEX group. The ectropion of cell phosphatidylserine (PS) was detected by flow cytometry after stained with FITC-anti-annexin V mAb/PI. The changes of delta psi m and CL content during cell apoptosis were detected by DiOC6(3)/PE-anti-annexin V mAb and NAO/PE-anti-annexin V mAb, respectively. RESULTS: 6 hours after SNAP treatment, the thymocytes exhibited typical cell apoptotic characteristics and crenation appeared in most annexin V-positive cells. In DEX group, the delta psi m decreased and the rate of non-apoptotic cells was significantly higher than that in blank control group, but there was no significant difference between SNAP and control groups. 40%-50% of DiOC6(3)-negative cells in each group had the same size as normal cells. The percentage of the cells with reduced CL content in SNAP group was significantly higher than that in blank control group (P<0.01). Non-apoptotic cells with reduced CL content were not found. In blank control group and SNAP group, percentage of normal-sized cells with reduced CL was (48.32+/-3.96)% and (43.64+/-4.90)%, respectively. CONCLUSION: The process of thymocyte apoptosis mediated by NO in mice includes successively PS ectropion, mitochondrial depolarization, CL oxidation and cell crenation. As compared with DEX group, the mitochondrial change mediated by NO is a later event in cell apoptosis.

[Study on the mitochondrial inner membrane potential of apoptotic thymocytes at cell and organelle level].

In this study, dexamethasone was used to induce mouse thymocyte apoptosis. The PI and Annexin V/PI staining flowcytometry methods were adopted to detect late and early phases apoptosis. Inner mitochondrial membrane potential (deltapsim) was examined by JC-1 and DiOC6(3)/PI staining flowcytometry. Direct JC-1 staining technology was applied to test the of deltapsim abstracted pure mitochondria. Results showed: DEX could significantly induce late and early phase mouse thymocyte apoptosis; at cell level, DEX was observed to reduce staining ability of deltapsim dependant fluorescence, J-aggregate and DiOC6(3), to drop down mitochondria number, but to cause no significant change of cell membrane integrity. Results of pure mitochondria detection showed most of them maintained normal deltapsim. According to above results, we concluded DEX could reduce mitochondria number when inducing mouse thymocyte apoptosis; and the remaining ones maintain normal function to meet the energy need for apoptotic process.

[Change of mitochondria during apoptosis of HL-60 cells induced by camptothecine].

AIM: To study the changes of mitochondrial mass and membrane potential (deltapsi(m)) during apoptosis of human promyelocytic leukemia cells (HL-60) induced by camptothecine(CPT). METHODS: HL-60 cells were stimulated with 4x10(-6) mol/L CPT. Apoptosis and necrosis of HL-60 cells were detected by annexin V-FITC/PI staining. Mitochondrial mass and membrane potential (deltapsi(m)) were measured by NAO and DiOC(6)(3) stanings, respectively. RESULTS: After 12 hour treatment with 4x10(-6) mol/L CPT, the early apoptotic rate of HL-60 cells was significantly increased compared with the control group (12.75+/-4.61)% vs (2.88+/-2.49)%,(P<0.01), and the necrotic rate was significantly elevated too, (3.48+/-1.67)% and (0.71+/-1.10)%, (P<0.01). The result of PI staining showed that the apoptotic rate of HL-60 cells at late phase (12 h) in CPT group was (3.52+/-1.07)%, whereas that in control group was (0.46+/-1.06)%. At the same time, we observed that the cells in G(2)/M phase were arrested. The percentage of cells in G(2)/M phase in CPT group and the control was (13.45+/-1.91)% and (22.46+/-2.19)% (P<0.01) respectively. The percentage of cells with low mitochondrial mass in CPT and control groups was (25.74+/-2.09)% and (4.53+/-1.26)%, respectively (P<0.01). Mitochondrial membrane potential in CPT group and control group was (17.71+/-5.23)% and (1.64+/-2.00)%, respectively. CONCLUSION: During CPT-induced apoptosis of HL-60 cells, mitochondrial mass and membrane potential drop significantly, but its depolarization heightens.

[The effect of p38 on the cycloheximide-induced HL-60 cell death through mitochondria pathway].

OBJECTIVE: To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway. METHODS: Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points. RESULTS: The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01). CONCLUSION: CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

[Cloning, sequencing and bioinformatics analysis of a new tumor suppressor gene ndr2 from mouse].

BACKGROUND & OBJECTIVE: Ndr2 (N-myc down stream regulator) gene in human is a new gene cloned with the human adult whole brain cDNA as template in 1999, which accession number is AF159092 in GenBank. Locating backward position of the N-myc gene in human chromosome, this gene was named Ndr2 gene. The previous experimental results showed Ndr2 gene probably is a tumor suppressor gene. To research the function of Ndr2 gene, the authors cloned the genomic sequence of ndr2 from mouse. METHODS: To clone Ndr2 genomic sequence by reverse transcription-polymerase chain reaction(RT-PCR) with the mouse genome library as template; automatic sequencing was performed using 310 Genetic Analyzer; homogeneous analysis was made using GenBank BLAST; open reading fragment(ORF) analysis was made using PC Gene and ORF Finder; domain analysis was made using ProDom system. RESULTS: A fragment (about 3310bp,identified by agarose gel electrophoresis) was obtained using RT-PCR with the mouse genome library as template. The fragment was cloned in pMD18-T vector. BLAST analysis showed that the sequence was highly homogeneous (with the homogeneity rate of 91.4%) with Ndr2 gene in human and non-homogeneous with genomic sequence database in mouse. ORF analysis showed that there was a complete coding region in it, which including 8 extrons and 7 introns; it can interpret a protein containing about 200 amino acid residuals. ProDom analysis showed there was a domain like acyl carrier protein(ACP) in it. CONCLUSION: The authors cloned Ndr2 gene in mouse and proved that the sequence is a new genome sequence in mouse genomic sequence database. At present, the genome sequence has been submitted to GenBank(the accession number: AY151387).