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Biomedical subjects

J Arechaga

Publications and source records attributed to J Arechaga.

At least 19 recordsLinked to original sources
PubMed2000

The role of cleavage of cell structures during apoptosis.

In this work we have studied the behavior of some cell structures, such as actin, tubulin and chromatin during apoptosis induced in F9 cells after retinoic acid treatment. In this experimental model, all defined steps of morphological changes described for apoptosis are observed. The correlation between a partial maintenance of F-actin and microtubular structures and the spatial distribution of F-actin suggests a possible relationship between this molecule and the characteristic shape changes observed in apoptosis. Additionally, the disposition of monomeric G-actin suggests a possible relationship between the fragmentation of this molecule and the cleavage of DNA. The analysis of the U2af1-rs1 specific sequence shows that the internucleosomal fragmentation observed in this gene is randomly produced during apoptosis and is not dependent of demethylation status. The results obtained confirm that specific cleavage of these cell structures is inherent to the development of the apoptotic process and do not exclude the possibility that proteolysis of key actin and/or tubulin molecules or the cleavage of specific chromatin sequences other than the ones analyzed here, could control the different phases of the apoptotic process.

Actins↗
PubMed1997

Comparative analysis of in vitro development of outbred mouse embryos cultured in Krebs-Ringer or tyrode-derived media.

Culture media for mouse preimplantation development are usually derived from two basic solutions: Krebs-Ringer and Tyrode. We have used outbred mice (OF1) to make a comparative analysis of derived media of both types and their success in sustaining development from the one-cell to the blastocyst stage. The best results (up to 50%), in terms of overcoming the two-cell block and sustaining development to expanded blastocyst, were obtained with media derived from Tyrode's solution and with Krebs-Ringer-based media in which the high concentrations of certain metabolites (Cl-, K+, Mg2+ and PO(4)3+) were reduced to the values present in the Tyrode-derived media. Interestingly, medium T6 (based on Tyrode and incapable of avoiding developmental block on its own), yielded the best development rate with the addition of ethylenediaminetetraacetic acid (EDTA). The addition of glutamine to medium T6 did not have any effect. We further developed T7, a medium based on T6 but incorporating the concentrations of nutrients present in the oviduct. This medium also proved better than its Krebs-Ringer counterpart (MTF), but not better than T6, probably due to the different nutritive requirements in the transition from morula to blastocyst. The two most significant findings were: i) that Tyrode-based formulations were superior to media based on Krebs-Ringer solution, and ii) that medium T6 with EDTA was as effective for mouse egg culture as the recently developed KSOM medium.

Animals↗
PubMed1996

The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering.

The turbidity of a suspension of cell nuclei isolated from animal tissue homogenates is a complex case of non-Rayleigh scattering. As a first approximation to this system, we have characterized a number of factors that may contribute to the observed turbidity: cation-dependent chromatin condensation, thermal denaturation of chromatin, nuclear shrinking, and changes in the optical properties of the membrane bilayer. Small differences in cation concentration, particularly in the case of divalent cations, lead to large changes in chromatin supramolecular organization, thus to large turbidity effects; thermally-induced changes in turbidity have a similar origin, although they are less pronounced. Under certain circumstances, either salts or heat may induce condensation of chromatin, the latter being connected to the inner side of the nuclear envelope, nuclear shrinking ensues, and this in turn modifies the suspension turbidity. Finally, changes in the physical properties of the lipid bilayers or of the phospholipids in the nuclear envelopes may also have significant effects, though smaller than chromatin changes, in the overall turbidity of the nuclear suspension.

Journal Article↗
PubMed1996

Parthenogenetic activation of mouse oocytes using calcium ionophores and protein kinase C stimulators.

Fertilization involves the production of inositol trisphosphate and diacylglycerol with a subsequent increase in intracellular calcium concentration ([Ca2+]i) and the activation of a calcium-dependent protein kinase, the so-called protein kinase C (PKC). Methods of parthenogenetic activation have focused on this calcium wave which seems to be large enough to generate all the responses associated with fertilization and even finally inducing the activation of PKC activity. The specific stimulation of PKC by phorbol esters in turn elicits [Ca2+]i oscillations although no reports exist claiming that the mere activation of this protein is capable of sustaining embryonic development. In this paper we describe the effect of different calcium ionophores and phorbol esters as parthenogenetic agents on mouse oocytes compared with ethanol as the standard procedure. Phorbol esters (OAG) fail to activate a significant number of oocytes, with very few reaching blastocyst stage. However, when a calcium ionophore (A23187) is added, the percentage of embryos reaching the blastocyst stage increases to such an extent that it is the best chemical method assayed to date. We conclude that incubation with both compounds combined inhibits feed-back processes between the above reactions and so induces a more physiologic parthenogenetic activation.

Animals↗
PubMed1995

ENDO A cytokeratin expression in the inner cell mass of parthenogenetic mouse embryos.

During the preimplantation period of development, the first cellular polarization and diversification of the mouse embryo occurs. This process starts at the eight-cell stage and is directly driven by the cytoskeleton. Cell polarization finally leads to the first embryonic epithelium, the trophectoderm, characterized by the presence of cytokeratins. It has not been described whether genomic imprinting, an epigenetic modification of certain genes depending on the parent-of-origin, affects preimplantation development. However, implantation is one of the steps in which an exceptionally high mortality rate is observed in mouse parthenogenetic embryos, a phenomenon that may be influenced by a deficiency in trophectoderm differentiation. To assess this possibility we analyzed the expression of various cytoskeletal proteins in late preimplanted embryos. No differences were observed in the expression of microtubules and microfilaments, but surprisingly, the undifferentiated cells of the parthenogenetic inner cell mass showed distinct cytokeratin staining. This anomalous cytoskeleton expression may be considered as one of the earliest manifestation described to date of the effect of genomic imprinting in development.

Animals↗
PubMed1994

Co-expression of laminin and a 67 kDa laminin-binding protein in teratocarcinoma embryoid bodies.

The synthesis of laminin chains is usually correlated to specific functions of laminin during embryo development. In this study we show that the CE44 teratocarcinoma embryoid bodies synthesize B1 and B2 chains of laminin as well as a 67 kDa laminin-binding protein while simultaneously differentiating into parietal endoderm. The intracystic presence of laminin and the 67 kDa cell surface laminin-receptor in teratocarcinoma differentiated cells suggest that the B chains of laminin play an important role in induction and/or mediation of cell differentiation and confirm the importance of laminin A chain in cell polarization and the supramolecular rearrangement of definitive basement membrane.

Animals↗
PubMed1988

Differentiation of ICM cells into trophectoderm.

It has been established previously that when inserted in the blastocyst E Ca 247 preferentially differentiates into trophectoderm in vitro. If the concept that tumors are caricatures of the process of tissue renewal is correct, then some cells from the inner cell mass (ICM), the normal counterpart of embryonal carcinoma, should be able to differentiate into trophectoderm. This has been a controversial issue. Four experiments are now reported that support the idea that ICM can differentiate into trophectoderm: 1) ICM from early blastocysts after classical immunosurgery made blastocysts in vitro; 2) ICM obtained from early blastocysts by immunosurgery using antigens other than histocompatibility ones made blastocysts in vitro; 3) ICM from early blastocysts, in which the trophectodermal cells had been labeled, contained no labeled cells following immunosurgery; and 4) In reconstruction experiments, polar and mural trophectodermal cells attached to ICM from late blastocysts failed to multiply and make blastocysts when cultured. It is concluded that like the embryonal carcinoma some ICM cells of early blastocysts have the potential to make trophectoderm. This fact is consistent with the concept that tumors are caricatures of the process of tissue renewal; and establishes E Ca 247 as a good model for study of trophectodermal differentiation.

Animals↗
PubMed1987

The fate of embryonal-carcinoma cells in mouse blastocysts.

Double-labeled embryonal-carcinoma (ECa) cells were injected into blastocysts or incorporated into blastocysts by aggregation, and their fate after various periods of time in culture was investigated. ECa-247 cells labeled with fluorescent microscopheres were easily identified in whole blastocysts. These blastocysts were embedded in plastic, serially sectioned, and prepared for autoradiography. The 3H-thymidine label on the embryonal-carcinoma cells allowed precise localization of the cancer-derived cells. ECa-247 cells preferentially localized in the mural trophectoderm, with a few being seen in primitive endoderm and, even more rarely, in the inner cell mass. Selected autoradiograms were re-embedded and thin sectioned for transmission electron microscopy. The cancer-derived cells were found to have differentiated in accordance with their localization.

Animals↗
PubMed1987

Morphometry of nuclear rings at high electron microscopic resolution.

In studying the fine structure of the nuclear chromatin, a new organelle was discovered, the nuclear ring. In its isolated form, this structure is rather like a geometric torus: a smooth, structureless ring. This paper presents the results of electron microscopic morphologic measurements on 424 isolated rings, i.e., rings found free of any fibrous connections. From the measurements of inner and outer diameters, other geometric features were also calculated. Overall, the size of the rings varied about fivefold. Inner and outer diameters were closely correlated, suggesting a rather stable thickness of the ring itself. The significance of the nuclear ring is as yet poorly understood. With its occasionally intimate connections to both chromatin fibers and to the inside of the inner nuclear envelope, its role is likely to be a crucial one. The membrane covering its opening is none other than the bilayer of the nuclear envelope. The concept of a patent opening or "pore" does not appear tenable in the face of this and related findings.

Chromatin↗
PubMed1986

Transglutaminase (TG) involvement in early embryogenesis.

Transglutaminase (TG) has been examined in different stages of preimplantation mouse embryogenesis. The specific activity of this enzyme in the soluble cellular fraction increases 2-fold from 2-cell embryos to 8-cell morulae and 4-fold from 2-cell embryos to blastocyst. The same developmental profile was seen when either N,N'-dimethylcasein or endogenous substrates were used in the TG assay. Using high-speed supernatants from different stage embryos as a source of enzyme and [3H]putrescine as acyl acceptor, the major acyl donor components were tubulin and a high molecular weight (HMW) cross-linkage product, as assessed by electrophoresis and immunoblotting. When either assembled or monomeric cytoskeleton proteins were compared as substrates, microtubules were the best acyl donors. These studies indicate that TG activity is modulated during the changing demands of blastomeres for microtubule cytoskeleton in early embryogenesis.

Animals↗
PubMed1983

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations.

To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.

Animals↗