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M Xing

Publications and source records attributed to M Xing.

33 records · Page 2Linked to original sources
PubMed1998

Immunolocalization of actin in intact and DNA- and histone-depleted nuclei and chromosomes of allium cepa.

The presence of actin in eukaryotic nuclei and chromosomes, and especially in higher plant nuclei and chromosomes, has not been well established. We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes, indicating that actin is present in the nuclei and chromosomes of the higher plant. We labeled sections of the meristematic cells of A. cepa with immunogold technique, gold particles were found over the whole nuclei and a number of gold particles were concentrated in condensed chromatin and nucleoli, confirming the results of the immunofluoresence observations. We treated the nuclei and chromosomes of A. cepa with DNase I and 2M NaCl and obtained DNA- and histone-depleted nuclei and chromosomes. Indirect immunofluorescence tests showed that the DNA- and histone-depleted nuclei and chromosomes reacted positively with the anti-actin antibodies. These results demonstrate that actin exists not only in intact nuclei and chromosomes, but also in DNA- and histone-depleted nuclei and chromosomes of the plant. In addition, our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A. cepa.

Actins↗
PubMed1997

Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways.

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

Animals↗
PubMed1997

Cystic fibrosis gene mutation (deltaF508) is associated with an intrinsic abnormality in Ca2+-induced arachidonic acid release by epithelial cells.

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells.

The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A2 (PLA2) activity in subsequently prepared MDCK-D1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA2 activity of cPLA2 immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA2 in MDCK-D1 cells. The BK-promoted increase of cPLA2 activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA2. However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKC alpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D1 cells (C. Godson, K.S. Bell, and P.A. Insel. [corrected] J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKC alpha-selective inhibitor GF109203X nor transfection of cells with PKC alpha antisense cDNA altered BK-mediated AA release. We conclude that PKC alpha is unlikely to play an important role in the regulation of cPLA2 by BK receptors in MDCK-D1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA2 activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D1 cells regulate cPLA2 via phosphorylation mediated by kinases other than ERK and PKC alpha.

Animals↗
PubMed1996

Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by alpha 1-adrenergic receptors in Madin-Darby canine kidney cells.

We have characterized the mechanism whereby a G protein-coupled receptor, the alpha 1-adrenergic receptor, promotes cellular AA release via the activation of phospholipase A2 (PLA2) in Madin-Darby canine kidney (MDCK-D1) cells. Stimulation of cells with the receptor agonist epinephrine or with the protein kinase C (PKC) activator PMA increased AA release in intact cells and the activity of PLA2 in subsequently prepared cell lysates. The effects of epinephrine were mediated by alpha 1-adrenergic receptors since they were blocked by the alpha 1-adrenergic antagonist prazosin. Epinephrine- and PMA-promoted AA release and activation of the PLA2 were inhibited by AACOCF3, an inhibitor of the 85-kD cPLA2. The 85-kD cPLA2 could be immunoprecipitated from the cell lysate using a specific anti-cPLA2 serum. Enhanced cPLA2 activity in cells treated with epinephrine or PMA could be recovered in such immunoprecipitates, thus directly demonstrating that alpha 1-adrenergic receptors activate the 85-kD cPLA2. Activation of cPLA2 in cell lysates by PMA or epinephrine could be reversed by treatment of lysates with exogenous phosphatase. In addition, both PMA and epinephrine induced a molecular weight shift, consistent with phosphorylation, as well as an increase in activity of mitogen-activated protein (MAP) kinase. The time course of epinephrine-promoted activation of MAP kinase preceded that of the accumulation of released AA and correlated with the time course of cPLA2 activation. Down-regulation of PKC by overnight incubation of cells with PMA or inhibition of PKC with the PKC inhibitor sphingosine blocked the stimulation of MAP kinase by epinephrine and, correspondingly, epinephrine-promoted AA release was inhibited under these conditions. Similarly, blockade of MAP kinase stimulation by the MAP kinase cascade inhibitor PD098059 inhibited epinephrine-promoted AA release. The sensitivity to Ca2+ was similar, although the maximal activity of cPLA2 was enhanced by treatment of cells with epinephrine or PMA. The data thus demonstrate that in MDCK-D1 cells alpha 1-adrenergic receptors regulate AA release through phosphorylation-dependent activation of the 85-kD cPLA2 by MAP kinase subsequent to activation of PKC. This may represent a general mechanism by which G protein-coupled receptors stimulate AA release and formation of products of AA metabolism.

Animals↗
PubMed1996

P2-purinoceptors utilize multiple signalling pathways in MDCK-D1 cells.

1. Madin-Darby canine kidney (MDCK) cells are a widely used model system for the study of epithelial cells. We have utilized a clonal variant, MDCK-D1, to examine signalling by P2-purinoceptors. 2. Several lines of evidence that lead us to conclude that MDCK-D1 cells co-express P2a- and P2y-purinoceptors and that both subtypes are linked to the release of arachidonic acid and metabolites (AA) include: (a) relative potency of nucleotide analogues in promoting AA release; (b) blockade by the antagonist suramin of response to the P2Y-selective agonist, 2-methylthio ATP (2-MT-ATP), but not to the P2a-selective agonist, UTP; and (c) additivity of response to 2-MT-ATP and UTP. AA release is a consequence of activation of phospholipase A2 (PLA2), most likely the 85 kDa cytosolic PLA2. 3. Treatment of MDCK-D1 cells with ATP, but not UTP, increases inositol 1,4,5-trisphosphate formation while both UTP and ATP increase phosphatidylcholine hydrolysis, ATP, UTP, and 2-MT-ATP can also stimulate phospholipase D activity. 4. Purine nucleotides increase cellular cAMP levels in MDCK-D1 cells in a manner that depends, at least in part, on activation of cyclooxygenase, since cAMP generation stimulated by ATP or UTP is inhibited by treatment of cells with indomethacin. Because cyclooxygenase-derived PGE2 can bind to prostaglandin receptors and stimulate synthesis of cAMP, nucleotides may raise cAMP in an autocrine or paracrine fashion. 5. Taken together, these results indicate that MDCK-D1 cells co-express P2a and P2y-purinoceptors and that these receptors utilize several mechanisms to regulate cell function, including activation of multiple phospholipases and autocrine/paracrine action of products.

Adenosine Triphosphate↗
PubMed1996

Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells.

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.

Adenosine Triphosphate↗
PubMed1995

Arachidonic acid release from NIH 3T3 cells by group-I phospholipase A2: involvement of a receptor-mediated mechanism.

Group I pancreatic phospholipase A2 (PLA2 I) is primarily a digestive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLA2 I binding to its receptor induces cellular chemokinesis, proliferation, and smooth muscle contraction. This enzyme also induces the production of prostaglandin E2 in certain cells and may have a proinflammatory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA2-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA2-I is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve of PLA2-I-induced AA release was saturable with an EC50 of 14.01 +/- 1.36 nM (n = 3). [3H]-AA was preferentially released over [3H]-oleic acid by PLA2-I. PLA2-I, inactivated with 4-bromophenacyl bromide, was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA2-I-induced AA release in NIH-3T3 cells. This release of AA is not dependent on protein kinase C or Ca2+ concentration. Comparison of the effect of PLA2-I with those of ATP and platelet-derived growth factor indicates that each of these agonists regulates AA release via independent pathways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA2 nor the protein level of this enzyme was affected by treatment of cells with PLA2-I. However, the increase in basal enzymatic activity of 85 kDa PLA2 due to protein kinase C activation was further enhanced by pretreatment of cells with PLA2-I. We conclude that: (1) short-term exposure of cells to PLA2 I does not cause measurable AA release; (2) release of AA from intact cells by this enzyme requires long-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA2 I; and (4) AA release by PLA2 I is accomplished via a receptor-mediated process. Taken together, these results raise the possibility that PLA2 I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/or to initiate new ones when chronic exposure of cells to this enzyme occurs.

3T3 Cells↗
PubMed1995

[Study of operative indication and timing for acute necrotizing pancreatitis].

65 patients of acute necrotizing pancreatitis were reported. 36 patients underwent operation with a mortality of 27.8%. However the mortality in 29 patients treated nonoperatively, the mortality was 3.4% because of their less critical condition. In operation group 7 out of 17 patients died (41.2%) with positive results in bacterial smear or culture while all 5 patients with negative results survived. The time from onset to admission in the survivor and the dead was 33 hours and 85 hours respectively. The time from onset to operation was 41 hours in the survivor while 138.8 hours in the dead. So the prognosis related closely to early intensive care therapy and if indicated, surgical intervention timely. The indication and timing of operation were discussed.

Acute Disease↗
PubMed1994

Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like HL60 cells. Linkage between impaired responses to agonists and absence of protein kinase C-dependent phosphorylation of cytosolic phospholipase A2.

We compared the regulation of cytosolic phospholipase A2 (cPLA2) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca(2+)-mobilizing P2-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P2-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA2 and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca2+, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA2. The stimulatory effects of GTP gamma S and Ca2+ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA2 coupling. Basal and PMA-stimulated phosphorylation of cPLA2 was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA2 phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.

Adenosine Triphosphate↗
PubMed1994

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa.

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G2--early prophase. The 250-400 nm thick compact chromatin threads in G2 nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1-1.5 microns) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120-250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.

Allium↗
PubMed1992

Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes.

We studied the regulation of arachidonic acid (AA) release by guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) and Ca2+ in electropermeabilized HL60 granulocytes. Stimulation of AA release by GTP gamma S and Ca2+ was mediated by phospholipase A2 (PLA2) and required the presence of MgATP (EC50: 100-250 microM). The nucleotide effects were Ca(2+)-dependent (maximal effects detected at 1 microM free cation). UTP and ATP gamma S, which stimulate AA release in intact HL60 granulocytes with potencies and efficacies similar to those of ATP, were ineffective in supporting the effects of GTP gamma S in electropermeabilized cells. Pretreatment with pertussis toxin affected stimulation of AA release by ATP in intact cell, without altering the nucleotide effects in permeabilized cells. We observed the protein kinase C-dependent phosphorylation of PLA2 in permeabilized HL60 granulocytes, together with a correlation between the effects of phorbol esters and staurosporine on this reaction and on AA release. ATP-independent activation of PLA2 by GTP gamma S and/or Ca2+ was measured in subcellular fractions prepared from HL60 granulocytes. These data appear consistent with a model in which PLA2 activity in resting HL60 granulocytes is subjected to an inhibitory constraint that prevents its activation by Ca2+ and G-proteins. Removal of this constraint, either by the protein kinase C-dependent phosphorylation of the enzyme in vivo or physical disruption of the regulatory assembly (e.g. by N2 cavitation), allows its activation by Ca2+ and G-proteins.

Adenosine Triphosphate↗
PubMed1992

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.

Adenosine Triphosphate↗
PubMed1991

[An electron microscopic study on the RNA component of synaptonemal complexes in spermatocytes of Mus musculus].

The ultrastructural and cytochemical features of synaptonemal complexes (SC) in sections of spermatocytes of Mus musculus were studied under electron microscope. In specimens stained with uranyl acetate and lead citrate the SC was found consisting of three main elements. the lateral element (LE), the central element (CE) and the transverse filament (L-C filament). When stained with the Bernhard's technique, the SC was recognized as a contrasted, tripartite structure which was usually located in the bleached area occupied by the condensed chromatin and composed of highly electron-dense LEs and medium electron-dense CE and L-C filaments. The SC and the LE, stained either by uranyl acetate-lead citrate or by the Bernhard's technique, always showed diameters of about 210 nm and 60 nm, respectively. The results suggest that RNA may be an important component of the SC.

Animals↗