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I Meza

Publications and source records attributed to I Meza.

At least 55 records · Page 3Linked to original sources
PubMed1983

Isolation and characterization of actin from Entamoeba histolytica.

Actin has been identified and purified partially from trophozoites of Entamoeba histolytica HMI-IMSS by a procedure that minimizes proteolysis. In cellular extracts, Entamoeba actin would copolymerize with muscle actin, but would not bind to DNase I or form microfilaments. Fractionation of the extracts by DEAE-cellulose and Sephadex G-150 chromatography yielded a purified actin that would copolymerize with rabbit skeletal muscle actin or polymerize alone into long filaments at 24 degrees C upon addition of 100 mM KC1 and 2 mM MgCl2. These filaments are not cold-stable and will depolymerize at 4 degrees C in 1 or 2 h. Entamoeba actin filaments bind phallotoxin with the same affinity as muscle actin and decorate with rabbit skeletal muscle heavy meromyosin. Entamoeba actin filaments activate the Mg2+ ATPase of heavy meromyosin to the same Vmax as muscle actin, but the Kapp is 2.8 times higher. Entamoeba actin is a single species with a slightly higher molecular weight than muscle actin (45,000) and a more acidic pI (5.4). The purified actin does not bind to DNase I, produce inhibition of the enzymatic activity, or block the binding of muscle actin. Comparison of the peptides obtained by limit digest with protease V8 from Staphylococcus aureus shows sequences with common mobility between alpha-actin and Entamoeba actin, but additional peptides are present which may account for the different properties of the Entamoeba actin. Finally, in vitro translation of mRNA from trophozoites produces a single polypeptide equivalent to the molecule purified from Entamoeba extracts.

Actins↗
PubMed1983

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes.

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.

Actins↗
PubMed1982

Occluding junctions in MDCK cells: modulation of transepithelial permeability by the cytoskeleton.

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In additions we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of greater than or equal to 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.

Animals↗
PubMed1981

Fluxes, junctions, and blisters in cultured monolayers of epithelioid cells (MDCK).

The formation of blisters is a transient phenomenon that led Dr. Leighton to describe its observation with time-lapse photography in the MDCK monolayer to a "gently boiling oatmeal." One may ask why is it transient and why most areas are momentarily not blistering. The observations discussed in this article indicate that (a) at the blister, junctions are really tight; (b) when transported fluid is allowed to escape through a permeable support, junctions are also tight, but (c) when the support is impermeable the junctions allow ouabain and peroxidase through. This suggests that, if the attachment of the monolayer is strong enough, the accumulation of fluid (Figure 7) bursts the junctions. If, on the contrary, junctions withstand the pressure, factor 3 of Figure 7 prevails over the others, and a blister is formed. In all the rest of the monolayer junctions seem to be open. This is in keeping with the observation by Rabito et al.,27 that if the monolayers are prepared on weakened supports, blisters are much bigger than under control conditions. It also allows the measurement of ionic fluxes and labeling of pumping sites at the basolateral membrane. As a corollary, one may say that some of the factors known to affect blistering 26 may very well act through modifications in the occluding junctions.

Animals↗
PubMed1981

Occluding junctions in cultured epithelial monolayers.

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80-600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.

Animals↗
PubMed1980

Structural and functional membrane polarity in cultured monolayers of MDCK cells.

MDCK cells form monolayers which have many of the properties usually found in transporting epithelia. The present article is devoted to the study of the structural and functional polarization of MDCK cells, which is one of the central features of transporting epithelia. The results show: (i) that MDCK monolayers transport 2.6 mumol hr-1 cm-2 of sodium in the apical to basolateral direction; (ii) the passive flux of this ion is relatively large (20.3 mole hr-1 cm-2), which is a characteristic of leaky epithelia; (iii) a large fraction of the penetration of sodium into the cells proceeds through an amiloride-sensitive channel, and the exit is operated mainly by a ouabain-sensitive pump; (iv) the net transport of sodium from the apical to the basolateral side agrees with the asymmetric labeling of the pumps with 3H-ouabain; (v) this asymmetric labeling agrees, in turn, with a higher concentration of intramembrane particles (IMPs) in freeze-fracture replicas of the basolateral side of the plasma membrane; (vi) the structural polarization of confluent MDCK cells is also revealed by the location of microvilli, occluding junctions, and pinocytotic vesicles; and (vii) the presence of a continuous ring formed by actin microfilaments visualized by immunofluorescence under the lateral aspect of the plasma membrane that may be related to the distribution of the occluding junctions, which act as barriers separating apical from basolateral membrane components.

Amiloride↗
PubMed1980

Experimental modulation of occluding junctions in a cultured transporting epithelium.

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.

Animals↗
PubMed1980

Occluding junctions and cytoskeletal components in a cultured transporting epithelium.

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible. The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTA-opened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

Animals↗
PubMed1978

Translation "in vitro" of globin mRNA from Xenopus laevis.

RNA isolated from Xenopus laevis reticulocytes and characterized as globin mRNA (Meza et al., 1978) was tested for its capacity to stimulate "in vitro" a wheat germ translation system, and the ability to synthesize a polypeptide. The latter was identified as globin by its electrophoretic mobility and immunoprecipitation with antiglobin antibody.

Animals↗
PubMed1978

Characterization of globin mRNA from Xenopus laevis.

A 9S polyadenylated mRNA was isolated from 'xenopus laevis reticulocytes and purified by sedimentation and oligo-dT-cellulose chromatography. The size of this RNA estimated in denaturing agarose gels was 2.2 x 10(5) daltons. 9S polyadenylated mRNA hydridized to total DNA with a Cot1/2 of 3.8 x 10(3).

Animals↗
PubMed1973

The mechanism of action of colchicine. Colchicine binding properties of sea urchin sperm tail outer doublet tubulin.

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50 degrees C (t((1/2)) = 5.4 min) and incubation with colchicine at 37 degrees C in the presence of vinblastine sulfate (t((1/2)) = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 +/- 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10(5) liters/mol at 37 degrees C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K(i) = 1.3 x 10(-6) M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37 degrees C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.

Animals↗