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I Meza

Publications and source records attributed to I Meza.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

Fibronectin "receptor" in Entamoeba histolytica: purification and association with the cytoskeleton.

Preferential binding of E. histolytica trophozoites to fibronectin (FN) is mediated by a 37 kDa peptide (1). We report now the further purification of this "putative" FN receptor (FN-R) and its characterization as an external membrane component also associated to the cortical cytoskeleton. The FN-R was solubilized from Triton-cytoskeletons and separated by chromatography in FN-Sepharose. The FN-R, labeled when live trophozoites were iodinated with 125I, remained associated to the cytoskeleton together with the FN bound to it, but was removed from the surface by trypsinization. The association of the FN-R to the cytoskeleton was inhibited by cytochalasin D, which also interferes with the adhesion of trophozoites to FN substrates and the organization of actin adhesion plates.

Actin Cytoskeleton↗
PubMed1991

Chromatin organization in Entamoeba histolytica.

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.

Actins↗
PubMed1990

Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica.

A 125-kD surface antigen of Entamoeba histolytica is recognized by 73% of immune sera from patients with amoebic liver abscesses. Using pooled human immune sera a cDNA clone (lambda cM17) encoding this antigen (M17) has been isolated from a lambda gt11 expression library of the virulent stain E. histolytica HM1:IMSS. Monospecific antibodies, purified by binding to phage lysate of lambda cM17, and mAb FA7 reacted exclusively with the 125-kD antigen by Western blot analysis. Surface binding and cap formation are observed with patient sera, purified monospecific antiserum, and mAb FA7. Corresponding genomic clones (pBSgM17-1/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp, which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with lambda cM17. The inferred amino acid sequence predicts a 125,513 dalton protein that contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic NH2-terminal region may serve as membrane anchor or signal sequence. In contrast to conservation of an immunodominant epitope recognized in pathogenic and nonpathogenic strains by monoclonal FA7 and human immune sera, amplification and sequence analysis of a 1,4000-bp fragment of this gene from a fresh nonpathogenic isolate by use of the PCR demonstrate regions of significant sequence divergence in this antigen. A 1% sequence variability among different isolates of the pathogenic strain HM1:IMSS and a 12-13% variability between pathogenic and nonpathogenic strains are revealed by comparison to published partial amino acid sequences (Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Proc. Natl. Acad. Sci. USA. 86:5118). Some restriction enzymes were found that allowed PCR diagnosis of nonpathogenic and pathogenic isolates with the exclusion of E. histolytica-like Laredo, suggesting that a detailed study of nonpathogenic and pathogenic isolates in relation to the M17 antigen sequence will provide a basis of differentiating isolates.

Amebiasis↗
PubMed1989

Synthesis and phosphorylation of cytoskeletal proteins during the in vitro biogenesis of MDCK cell monolayers.

In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconfluent and early confluent cells. On the other hand a decrease in total protein, actin, vimentin and cytokeratin synthesis was observed in cells kept in suspension for 24 h or in 78-h or 174-h older confluent cultures. These cultures also showed a decrease in the uptake of labeled amino acid. Cytokeratin and vimentin phosphorylation rates were also modified during the in vitro formation of a monolayer. In suspended cells, cytokeratins were phosphorylated and three labeled isoelectric variants of the 40, 48 and 58K (K = 10(3) Mr) cytokeratins were present in intermediate filament extracts. In subconfluent and early confluent cells only two isoelectric phosphorylated variants of the 40, 48 and 58K cytokeratins were detected and vimentin was also phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1989

Cerebral infarction associated with cocaine use.

We report the case of a young man with an acute infarction of the left putamen and caudate nucleus, whose symptoms appeared six hours after intranasal use of approximately 0.5 g of cocaine hydrochloride. It seems probably that in this patient cocaine consumption played a role in the development of stroke.

Adult↗
PubMed1988

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization.

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.

Actins↗
PubMed1987

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.

Actins↗
PubMed1987

Isolation of a 220-kilodalton protein with lectin properties from a virulent strain of Entamoeba histolytica.

A 220-kilodalton (kDa) protein with lectin properties was isolated from Entamoeba histolytica strain HM1:IMSS and was purified by Sepharose 4B chromatography and electroelution from 5% SDS-polyacrylamide gels. The protein contains 9% carbohydrate by weight; is rich in hydrophobic residues; and is very immunogenic in mice, hamsters, and rabbits. The protein binds to fixed monolayers of MDCK cells and inhibits trophozoite attachment to the cultured cells. The 220-kDa protein agglutinates human erythrocytes, and agglutination is inhibited by micromolar concentrations of hyaluronic acid, chitin, chitin-derived products (chitotriose), and antibodies to the purified protein. The 220-kDa protein is recognized by an antibody to the membrane but not by antibodies to other subcellular fractions. We therefore suggest that this 220-kDa protein with lectin properties is a component of the plasma membrane and could be one of the putative "receptor" molecules involved in cell and/or matrix attachment.

Adhesiveness↗
PubMed1987

Use of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolytica.

Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4B chromatography of Entamoeba histolytica strain HM38:IMSS homogenates, and the protein was found to have lectin properties. The antibodies specifically recognized this protein in trophozoite homogenates. Immunologically related molecules with the same molecular weight were identified by polyclonal antibodies in strains HM1:IMSS, Entamoeba invadens, and Entamoeba histolytica Laredo. Six monoclonal antibodies recognized only the 220-kDa protein present in strains HM38 and HM1, a result indicating the presence of similar epitopes in the proteins from virulent strains isolated from humans. All the antibodies against the 220-kDa protein have the following properties: (1) they bind to the plasma membrane of live or fixed trophozoites, (2) they partially inhibit the adhesion of trophozoites to erythrocytes and cultured cells, and (3) they inhibit erythrophagocytosis.

Adhesiveness↗
PubMed1986

A re-evaluation of cytoplasmic gelsolin localization.

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1986

Isoenzyme patterns of Entamoeba histolytica isolates from asymptomatic carriers: use of gradient acrylamide gels.

A vertical polyacrylamide gradient gel (3% to 7%) was designed to facilitate the electrophoretic resolution and classification of isoenzyme patterns of Entamoeba histolytica isolates. The following enzyme systems were used: phosphoglucomutase (PGM), hexokinase (HX), glucosephosphate isomerase (GPI), and malate dehydrogenase (ME). The modifications in the electrophoretic procedure and sample preparation allowed the reproducible comparison of enzyme patterns of axenic, monoxenic, and mixed cultures of E. histolytica isolated from humans. The clear distinction obtained in gradient polyacrylamide gels, between amebic isoenzyme bands and those from bacteria, renders this technique adequate for application to epidemiological studies where mixed cultures are used. The isoenzyme patterns of eight isolates from asymptomatic carriers, rigorously characterized by the absence of clinical, endoscopic, and serological findings were studied and compared with three well characterized pathogenic strains, cultured under axenic conditions. Our observations confirm the existence of distinct isoenzyme patterns for PGM, HX, and GPI in pathogenic and nonpathogenic strains, and reveal the consistent presence of more than one band for GPI. In addition, a previously undescribed band for GPI with an Rf of 0.64 in a carrier strain was found. The results suggest that while carriers usually harbor amebas with nonpathogenic isoenzyme patterns, pathogenic patterns also may be found in carriers.

Carrier State↗
PubMed1985

Isolation and partial characterization of human platelet vinculin.

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.

Actins↗
PubMed1985

Characterization of a 36,000-dalton protein from the surface of Madin-Darby canine kidney cells involved in cell attachment and spreading.

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.

Animals↗
PubMed1985

Mitogen-like monoclonal anti-actin antibodies.

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.

Absorption↗