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I Meza

Publications and source records attributed to I Meza.

At least 19 recordsLinked to original sources
PubMed2001

Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.

Animals↗
PubMed2000

Extracellular matrix-induced signaling in Entamoeba histolytica: its role in invasiveness.

Entamoeba histolytica trophozoites can invade the mucosa of the host large intestine to cause disease. Here, Isaura Meza describes recent work indicating that the trophozoites respond to the binding of extracellular matrix proteins by activating receptor-mediated signal transduction pathways. Activation of phosphokinase C or adenylyl cyclase induces protein phosphorylation, actin gene expression and reorganization of the actin cytoskeleton to form adhesive structures that promote interaction with the substrate and the release of proteases. The release of proteases at the sites of contact, with the subsequent degradation of the substrate and generation of chemotactic peptides, facilitates locomotion and dissemination of the invading trophozoites.

Adenylyl Cyclases↗
PubMed2000

Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

Actins↗
PubMed1998

Entamoeba histolytica: identification of functional Gs and Gi proteins as possible signal transduction elements in the interaction of trophozoites with fibronectin.

Trophozoites of Entamoeba histolytica adhere to several components of the extracellular matrix. Binding is mediated by specific receptors identified in the parasite surface. Interaction of trophozoites with FN induces the formation of special adhesion structures that are dynamic cytoskeleton membrane complexes and facilitate both adhesion and substrate degradation. The process requires activation of signaling pathways in which PLC, IP3, Ca2-, and PKC participate. These observations, and recent experiments showing increments in cAMP in the trophozoites during the interaction with FN, suggest that FN receptors in the amebic surface could be coupled to G-proteins. We report here that trophozoite plasma membrane peptides of 92, 49, 42, 37, and 21 kDa are ADP-ribosylated by Vibrio cholerae and Bordetella pertussis toxins. Three of them are also recognized by antibodies prepared against the alpha-subunit of Gs-and Gi-proteins. Adenylyl cyclase activity detected in isolated membranes was strongly stimulated by treatment with the toxins. Forskolin (an agonist of the enzyme) and FN also induced increments in the enzymatic activity. Live amebas incubated with the toxins showed enhanced adhesion to FN substrates and a striking reorganization of polymerized actin. The actin rearrangement is reminiscent of the one induced by either forskolin or dibutyril cyclic AMP treatment. Our present data show the presence and the functionality of Gs- and Gi-like proteins and their apparent activation during in vitro interaction of amebas with FN and complement previous observations indicating the operation of signal transduction mechanisms in E. histolytica.

Actins↗
PubMed1998

Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations.

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e < or = 5 microM) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin-myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10 min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction where Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid( an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin-myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]3 poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.

2,4-Dinitrophenol↗
PubMed1997

Fibronectin-derived fragments as inducers of adhesion and chemotaxis of Entamoeba histolytica trophozoites.

Active migration of Entamoeba histolytica trophozoites through extracellular matrixes might play a role in host tissue destruction. Trophozoites degrade soluble fibronectin (FN) bound to their surface and adhere to substrate-bound FN, producing local degradation. FN proteolytic fragments were used to determine the nature of adhesion and motility-promoting domains within the protein. The 70-kDa fragment (amino-terminal end) promoted the highest adhesion, followed by the 120-kDa fragment, which contains the cell-binding domain. The 25-kDa fragment (carboxy-terminal end of the A chain) promoted half the adhesion, while two Hep II-binding fragments had no effect. The 70- and 120-kDa fragments also stimulated directed migration and chemokinesis. Intact FN and the 25-kDa fragment showed lower stimulation. The Hep II-binding fragments had no activity. Results support previous evidence for distinct cell-surface components as mediators of adhesion to FN and trophozoite motility and the potential importance of cell matrix recognition and degradation in their invasive behavior.

Animals↗
PubMed1997

Myosin I interactions with actin filaments and trans-Golgi-derived vesicles in MDCK cell monolayers.

In MDCK cell cultured monolayers, as well as in natural and other cultured epithelia, the proper organization of the actin filament ring, tethered to the plasma membrane at the zonula adhaerens, is apparently necessary for their functioning as a transporting epithelium. It has been proposed that actin filaments, in conjunction with motor proteins, could provide the structural basis that regulates the tight junction (TJ) sealing capacity as well as the transport of membrane-tagged proteins required for cell polarization. To test this hypothesis, the authors analyzed the localization and possible association of the actin-binding motor protein myosin I with actin filaments during changes in the actin ring position and organization, and also with trans-Golgi-derived vesicles. Modifications of the ring were induced subjecting the cells to external Ca2+ depletion and restoration (Ca2+ switch), or by treatment with drugs known to depolymerize actin filaments (cytochalasin D, CD). The distribution of myosin I and actin, both in intact cells and in cellular fractions, was monitored using heterologous cross-reacting antibodies and phalloidin. The authors identified an isoform of myosin I of approximately 110-125 KDa, homologous to myosin IB of Acanthamoeba, a fraction of which colocalized with the peripheral actin ring. The association seems transient as, once the ring retracted as result of Ca2+ depletion, or became disorganized by CD, myosin not longer colocalized with the actin fibers but appeared dispersed in the cytoplasm. Furthermore, a significant fraction of the total myosin I in the cell was associated to Golgi-derived vesicles which could also associate in vitro with actin filaments. The authors' data support, then, the participation of myosin I, in association with actin filaments, in vesicle translocation to and from the cell membrane as proposed for natural epithelia, and provide a further insight into the structural organization that maintains epithelial cell polarity in cultured monolayers.

Actin Cytoskeleton↗
PubMed1996

Kinematic analysis of Toxoplasma gondii motility.

Toxoplasma gondii tachyzoites execute a complex and little understood combination of rapid movements to reach and penetrate human or other animals cells. In the present study, computer-assisted simulation was used to quantitatively analyze the motility of these parasites in three-dimensional space with spatial and temporal resolutions in the micrometer and subsecond ranges. A digital model based on electron-micrographs of a serially sectioned tachyzoite was animated according to a videomicrographed sequence of a characteristic repetitive movement. Keyframe animation defined over 150 frames by a total of 36 kinematic parameters for specific motions, of both the whole model and particular domains, resulted in a real-time life-like simulation of the videorecorded tachyzoite movement. The kinematic values indicate that a full revolution of the model is composed of three half-turns accomplished in nearly 5 s with two phases: a relatively slow 180 degrees tilting with regard to the substratum plane, followed by fast (over 200 degrees/s) spinning almost simultaneous with pivoting around the posterior end, each clockwise and for about 180 degrees. Maximal flexing of the body, as well as bowing and retraction of its anterior end, occur at midway during the tilting phase. An estimated 70 degrees. clockwise torsion of the body seems to precede the spinning-pivoting phase. The results suggest the operation of two basic forces in the motility of T. gondii tachyzoites: (1) a clockwise torque that causes torsion, spinning, and pivoting; and (2) a longitudinal pull that contracts, bends and tilts the parasite. We discuss the possibility that both of these forces might result from the action of an actin-myosin system enveloping the twisted framework of microtubules characteristic of these organisms.

Animals↗
PubMed1996

Fibronectin-induced intracellular calcium rise in Entamoeba histolytica trophozoites: effect on adhesion and the actin cytoskeleton.

The interaction of Entamoeba histolytica trophozoites with fibronectin (FN) promotes adhesion of the protein to the cells and its later degradation by locally released proteases. Binding to FN-covered surfaces induces, in addition, the formation of actin adhesion plates and focal contacts in the amebas. The signaling mechanisms underlying the response to FN are incompletely understood. In this paper we examined the modifications of cytosolic free calcium ([Ca2+]i) induced in the trophozoites by the interaction with FN and their effect on adhesion and the actin cytoskeleton organization. FN produced a sustained rise of [Ca2+]i that could be correlated to the incremented adhesion to FN-covered surfaces. Further increments in [Ca2+]i produced by Ca2+ ionophores A23187 or ionomycin significantly increased the adhesion of trophozoites, whereas depletion of cytoplasmic Ca2+, by treatment with the ionophores in the absence of external Ca2+ or using the chelator BAPTA/AM, blocked it almost completely. To study the role of internal calcium we used the plant lactone thapsigargin, which was found to produce a transient increase of [Ca2+]i but a low stimulatory effect on adhesion and the organization of actin plates. The shifting of soluble actin to the F-actin form and the stabilization of adhesion plates and focal contacts, seen as results, of the FN stimulus, were positively influenced by rises in [Ca2+]i and negatively affected by its decrement. Additional evidence for Ca2+ -mediated signaling in the response to FN was provided by the poor adhesion and defective actin plate organization observed in trophozoites treated with calmodulin antagonists. The results presented here suggest that FN action is mainly dependent on the influx of external Ca2+.

Actins↗
PubMed1996

Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes.

Swelling of cultured astrocytes exposed to hyposmotic medium modified the organization of the filamentous actin (F-actin) cytoskeleton, making the actin network diffuse in the cell body but concentrated at foci corresponding to the tips of the cell projections retracted by swelling. This change was reversible, and, after 2 h, the actin cytoskeleton tended to recover, and cells regained their flat and stellate shape. Cytochalasins B and D (CB and CD, respectively), which disrupt the actin cytoskeleton, did not affect regulatory volume decrease (RVD) or the swelling-activated efflux of Cl- and inositol, although 10 microM CD increased the basal efflux of taurine. The mercurial p-chloromercuribenzenesulfonate (0.5-1 mM), known to disrupt the membrane cytoskeleton in isosmotic conditions, induced a 46, 50, and 38% release of [3H]taurine, 125I, and [3H]inositol, respectively, causing cell shrinkage and retraction of the cytoskeleton. Coincidently, the swelling-stimulated release of [3H]taurine and 125I was reduced by 60 and 30%, respectively. Results of this study do not exclude the possibility that changes in the actin cytoskeleton elicited by swelling are involved in mechanisms of RVD and only indicate that the disruption caused by cytochalasins is unrelated to that process.

Actins↗
PubMed1995

Characterization of adhesion plates induced by the interaction of Entamoeba histolytica trophozoites with fibronectin.

Entamoeba histolytica trophozoites are pleiomorphic and highly motile cells. Although scarce fibrous material can be identified in the cytoplasm as elements of an organized cytoskeleton, clearly defined actin-containing structures are formed at the site of cell-matrix contact upon the interaction of trophozoites with fibronectin (FN) and other cellular matrix substrates. The structures are reminiscent of the adhesion plaques or focal contacts found in higher eukaryotic cells, where actin filament bundles insert into specialized regions of the plasma membrane and function as signal transduction organelles. Thus, the formation of adhesion plates in this parasitic ameba could be related to the specific signaling responses involved in its invasive behavior. Here, we report the isolation of amebic adhesion plates and the results of their structural and molecular analyses. Filaments, with the characteristic diameter of F-actin, radiating from an electron-dense matrix, are the main feature. Actin is one of the main protein components of the plate; other proteins identified are a FN-binding protein--previously reported as a "putative" FN receptor--the actin-binding proteins myosin II, myosin I, alpha-actinin, vinculin, and tropomyosin. The presence of the isolated plates of several proteases and protein kinases, in particular pp125FAK, is also demonstrated. our results suggest that adhesion plates in amebas are dynamic membrane-cytoskeletal complexes participating not only in the attachment to FN substrates but also providing the structural basis for their involvement in parasite locomotion and invasiveness.

Actins↗
PubMed1994

Entamoeba histolytica: PKC transduction pathway activation in the trophozoite-fibronectin interaction.

Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of the actin cytoskeleton and an increase in proteolytic activities that results in the degradation of the bound protein. The binding is mediated by a 37-kDa FN "receptor" localized in the trophozoite surface and associated to the cytoskeleton. The intracellular signals triggered by the ligand-receptor interaction are not well understood but it is plausible that they drive the observed responses. To address this issue, the activation of protein kinase C (PKC) pathways by FN binding was explored. Stimulation with phorbol myristate acetate (PMA) or FN produced a rapid increase in the amebas adhesion to the substrate and local release of proteases. Two PKC inhibitors, H7 and staurosporine, reverted the PMA stimulus and inhibited the response induced by FN. Interaction with FN as well as treatment with PMA produced transient changes of F-actin levels susceptible to inhibition by H7. Furthermore, phosphorylation of amebic proteins was enhanced in response to FN binding and PMA, while the presence of the PKC inhibitor diminished their phosphorylation. Inositol triphosphate production was stimulated by the FN binding, and PKC activation and translation was registered in cell extracts obtained from the stimulated amebas. Our results suggest that PKC pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba.

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome--coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3' end of the 26S rRNA--to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histolytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentiation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.

Animals↗
PubMed1992

Entamoeba histolytica: phylogenetic considerations. A review.

Comparison of 16S rRNA sequences alone positions E. histolytica in rRNA-based phylogenies branching after flagellates such as Euglenoids and Kinetoplastids, whereas morphological and functional features had suggested an earlier branching and different relationships. As several characteristics of this parasite do not precisely adjust to the phylogenetic frame obtained by comparison of ribosomal sequences, and the inferences from this approach could be skewed because of the high A+T content of Entamoeba rDNA, a re-evaluation of E. histolytica phylogeny seems convenient at this point. The data about genomic and protein sequences could provide bases to complement or expand the rRNA-based phylogeny.

Animals↗