Comparative study on effects of cytochalasins B and D on F-actin content in different cell lines and different culture conditions.

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.     

Latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D

The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament-mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G-actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two classes of drugs. To elucidate these differences we have compared the short- and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1-3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration-dependent changes in cell shape and actin organization. However, the latrunculin-induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 microgram/ml, latrunculin A caused complete rounding up of all cells at 0.2 microgram/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10-20 times higher. The short-term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long-term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B-induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E-115 cells.     

Alterations of the actin polymerization status as an apoptotic morphological effector in HL-60 cells

The alterations of the cytoskeletal actin network have been implicated as a morphological effector in apoptosis. However, studies directly linking actin change to the morphological events in apoptosis are lacking. This study quantitatively examined the effect of actin alteration on the camptothecin (CPT)-induced apoptotic process in HL-60 cells. Actin alteration was induced by two distinctive types of agent: the polymerization-stimulating agent, Jasplakinolide (Jas), and the polymerization-blocking agent, cytochalasin B (CB). The actin polymerization status was measured by two complementary methods: the cell pellet-based DNase I inhibition method, and the individual cell-based quantitative fluorescence image analysis (QFIA) assay. Actin polymerization induced by Jas caused apoptosis directly. By contrast, CB, an actin polymerization-blocking agent, partially inhibited CPT-induced apoptosis. A similar inhibition of the CPT-induced apoptosis response was observed with a more specific actin depolymerization agent, cytochalasin E. The alterations of the actin polymerization status occurred in three sequential steps during the apoptotic process: first polymerization, followed by depolymerization, and finally degradation. However, compared with CPT-induced apoptosis, Jas-induced apoptosis was characterized by pronounced actin polymerization that corresponded morphologically with prominent membrane blebbing, but less apoptotic body formation. Furthermore, DNase I activity, which is normally inhibited by G-actin, was specifically detected in Jas-treated cells. These results show that the regulation of actin polymerization is an important apoptotic morphological effector, whereas the alterations of the actin polymerization status by chemicals have profound effects not only on altering the morphology of apoptotic cells, but on apoptosis induction in HL-60 cells as well.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C

Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). The cytochalasin D-induced inhibition of SET activity was partially, but significantly, reversed by jasplakinolide, a cell permeable stabilizer of F-actin, whereas TPA-induced inhibition of SET activity was not reversed by jasplakinolide. To elucidate whether the subcellular localization of SET was changed in response to cytochalasin D or TPA, we expressed the SET fused with the green fluorescent protein (SET-GFP) in COS-7 cells and observed the subcellular distribution of SET-GFP under a confocal laser scanning fluorescent microscope. Neither cytochalasin D nor TPA markedly changed the SET-GFP cellular localization, although these drugs caused morphological change in the GFP-transfected COS-7 cells. In addition, SET activity was not altered by the treatment with either colchicine, an inhibitor of microtubule polymerization, or taxol, a stabilizer of microtubule polymerization. These results suggest that the SET uptake activity was regulated by the state of the actin cytoskeleton and that TPA exerts its inhibitory action on SET activity, in part, via disruption of F-actin and subsequent morphological change in cells.     

细胞松弛素D对棉铃虫核型多角体病毒复制和装配的影响

杆状病毒感染引起宿主细胞肌动蛋白骨架的构象变化 ,使之形成缆绳结构 .棉铃虫核型多角体病毒 (HaNPV)的衣壳蛋白也能使宿主昆虫的肌动蛋白发生凝聚 ,用细胞松弛素D抑制宿主肌动蛋白形成纤丝结构 ,病毒感染Hz AM1,空斑计数表明 ,0 1μg/ml细胞松弛素D可使棉铃虫核型多角体病毒的增殖下降 10 4倍 ,细胞松弛素D浓度增高到 0 5 μg/ml则测不到子代病毒粒子 .Western印迹分析表明 ,细胞松弛素D并不影响受染细胞中肌动蛋白的含量 .斑点印迹 (dotblot)也表明 ,病毒DNA的合成也没有受到影响 ,推测宿主细胞的肌动蛋白纤丝结构与病毒的复制有关 .在电子显微镜下观察超薄切片发现 ,在 0 5 μg/ml细胞松弛素D处理细胞中形成的病毒粒子形态与正常形态明显不同 ,提示细胞松弛素D抑制HaNPV的增殖是由于抑制病毒组装成完整有感染性的病毒粒子 .从而可以认为宿主昆虫细胞的丝状肌动蛋白对子代病毒的复制和组装是必需的 .     

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro‐inflammatory mediators. We examined mechanisms of LPS‐stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage‐like J774 cells. A wound‐healing assay revealed that LPS also activated directed cell movement that was followed by TNF‐α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5‐bisphosphate. Fractionation of Triton X‐100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin‐regulatory proteins, paxillin on tyrosine 118 by 80% and N‐WASP on serine 484/485 by 20%, and these events preceded activation of NF‐κB. LPS‐induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N‐WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS‐stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF‐α production suggesting that LPS‐induced cell motility is not required for TNF‐α release. J. Cell. Biochem. 113: 80–92, 2012. © 2011 Wiley Periodicals, Inc.     

Human T lymphocytes synthesize the 92 kDa type IV collagenase (gelatinase B)

In order for T cells to exit the circulatory system, traverse the endothelial basement membrane, and arrive in target tissues, these cells must attach to and degrade basement membrane proteins. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to stimulate lymphoid cell adhesion to basement membrane components. We have used TPA to study the ability of human lymphoid cells to secrete type IV collagenases, enzymes capable of degrading basement membrane proteins. Here, we found that human primary T cells and H-9 lymphoid cells synthesize the 92 kDa type IV collagenase (gelatinase B) and TPA stimulates the synthesis and secretion of this protease. Peak TPA-stimulated gelatinase B secretion and mRNA accumulation were observed 9 hours after TPA treatment, while the peak adhesion to type IV collagen was observed only 3 hours after TPA treatment. The protein kinase C inhibitor, H-7, inhibited TPA-stimulated gelatinase B secretion. Both the primary T cells and H-9 lymphoid cells also expressed the mRNA for the tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that TPA - stimulated lymphoid cells adhere to type IV collagen and subsequently synthesize and secrete gelatinase B and TIMP-1. We conclude that lymphoid cell extravasation may involve cellular employment of adhesion mechanisms prior to degradation of the matrix, which is similar to the process of extravasation used by metastatic cells. © 1993 Wiley-Liss, Inc.     

Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D-leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin beta3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin beta3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin alphaVbeta3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.     

The actin cytoskeleton controls the efficiency of killer Ig-like receptor accumulation at inhibitory NK cell immune synapses

Killer cell Ig-like receptors (KIRs) are MHC class I-specific receptors expressed in NK and T lymphocytes. KIR antagonism of activation signals occurs at the immune synapse between the effector and target cells. The processes that regulate clustering of KIR are not well defined. We have expressed KIR-GFP receptor chimeras in two human NK-like lines, YTS and NK92. In this study, we show that the frequency of KIR enrichment at the synapse was decreased for a KIR that lacks a portion of the cytoplasmic tail. Strikingly, blocking actin polymerization with a high dose of cytochalasin D also substantially decreased clustering of KIR as well as KIR-induced clustering of HLA-C-GFP in target cells. However, the effect of inhibiting actin polymerization was only clearly evident at the earlier time points after cell mixing, and eventually clustering of KIR and HLA-C occurred independently of actin remodeling. Although treatment with anti-LFA-1 also decreased conjugate formation, the frequency of KIR clustering remained normal within the population of conjugates that did form, suggesting that the effect of cytochalasin D is not solely through LFA-1. Collectively, these data suggest that the actin cytoskeleton and the cytoplasmic tail of KIR regulate the efficiency by which KIR accumulates at inhibitory NK cell synapses.     

The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells

To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor beta1 (TGFbeta1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFbeta1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFbeta1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFbeta1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFbeta1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFbeta1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.     

Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin

The cyclic heptapeptide hepatotoxin microcystin-LR from the cyanobacterium Microcystis aeruginosa induces rapid and characteristic deformation of isolated rat hepatocytes. We investigated the mechanism(s) responsible for cell shape changes (blebbing). Our results show that the onset of blebbing was accompanied neither by alteration in intracellular thiol and Ca2+ homeostasis nor by ATP depletion. The irreversible effects were insensitive to protease and phospholipase inhibitors and also to thiol-reducing agents, excluding the involvement of enhanced proteolysis, phospholipid hydrolysis, and thiol modification in microcystin-induced blebbing. In contrast, the cell shape changes were associated with a remarkable reorganization of microfilaments as visualized both by electron microscopy and by fluorescent staining of actin with rhodamine-conjugated phalloidin. The morphological effects and the microfilament reorganization were specific for microcystin-LR and could not be induced by the microfilament-modifying drugs cytochalasin D or phalloidin. Using inhibition of deoxyribonuclease I as an assay for monomeric actin, we found that the microcystin-induced reorganization of hepatocyte microfilaments was not due to actin polymerization. On the basis of the rapid microfilament reorganization and the specificity of the effects, it is suggested that microcystin-LR constitutes a novel microfilament-perturbing drug with features that are clearly different from those of cytochalasin D and phalloidin.     

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.     

Interaction of tumour promoters with epithelial cells in culture : An immunofluorescence study

12-O-tetradecanoyl phorbol-13-acetate (TPA) has a profound and rapid influence on the cytoskeleton of Madin-Darby Canine Kidney (MDCK) cells. Within 10 min, TPA induces a rapid change in morphology, from a flat, cuboidal state to a rounded or elongated morphology in which the cell membranes become convoluted. Concomitant with this morphological change is a rapid dissolution of stress fibres and a redistribution of F-actin from microfilament bundles to a membrane or sub-membranous location. The rearrangement of actin is paralleled by a rearrangement of alpha-actinin and a reduction in the number of vinculin-containing adhesion plaques. Unusual F-actin configurations are often found emanating from a perinuclear location, usually containing alpha-actinin and terminating in a vinculin-containing adhesion plaque. The cytoskeletal rearrangements occur in the presence of inhibitors of protein synthesis or oxidative phosphorylation, but do not occur if glycolysis is also inhibited. The rearrangements are partly abrogated by the presence of cytochalasin B (CB). Despite these dramatic changes in microfilaments the polymerization state of actin remained unaltered after TPA treatment. Furthermore, although changes in the movement of membrane lipids have been reported, no obvious differences in the ability of glycoproteins to redistribute in the plane of the membrane were found as judged by FITC-concanavalin A (conA) induced patching. The rapidity of the morphological response of MDCK cells to TPA indicates that the cytoskeleton is one of the primary targets of TPA, but that tumour promoters differ from RNA tumour viruses in their effect on the state of actin polymerization.     

Differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production

Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.     

LPS-induced lung injury in neonatal rats: changes in gelatinase activities and consequences on lung growth

Postnatal lung growth disorders may involve imbalance between metalloproteinases and their inhibitors. Inflammatory cell 92-kDa gelatinase overactivity has been reported in adults with lung injury but has not been looked for in neonates. We compared gelatinase activity in neonatal and adult rats and evaluated postnatal lung growth after lipopolysaccharide (LPS)-induced lung injury. Significant intra-alveolar inflammatory cell recruitment occurred in adults and neonates; cell counts increased 16-fold in adults and 2.7-fold in neonates. Total 92-kDa gelatinase activity was increased in neonates and adults and was significantly correlated to inflammatory cell counts. For a given cell count, 92-kDa gelatinase increased more in neonates than in adults. Morphometric neonatal lung analysis showed that LPS-injured lungs had decreases in absolute values of lung volume (P < 0.03), alveolar surface (P < 0.004), and air space volume (P < 0.03). Doxycycline, a nonspecific metalloproteinase inhibitor, partly inhibited LPS-induced 92-kDa gelatinase overactivity but did not improve LPS-induced alveolar growth disorders. LPS-mediated lung injury in neonatal rats induced both gelatinase B overactivity and alveolar growth disorders, although no causal link between these two effects was demonstrated.