Biomedical subjects

M Opas

Publications and source records attributed to M Opas.

At least 55 records · Page 3Linked to original sources

PubMed1992

Widespread tissue distribution of rabbit calreticulin, a non-muscle functional analogue of calsequestrin.

Calreticulin was identified in a variety of rabbit tissues by Western blot analysis. Indirect immunofluorescence studies on cultured cells or frozen sections from the corresponding tissues revealed that the protein was distributed to the endoplasmic reticulum or sarcoplasmic reticulum. Calreticulin was found to be an abundant calcium-binding protein in non-muscle and smooth muscle cells and a constituent calcium-binding protein in cardiac and skeletal muscle. From the immunoblot data, calreticulin may exist as an isoform in rabbit neural retina. The present study establishes the ubiquity of calreticulin in intracellular calcium binding.

Animals↗

PubMed1992

Effects of insulin and a tumour promoter, TPA, on glucose transport and metabolism in retinal pigmented epithelium in vitro.

We have studied the effects of insulin, adenosine and 12-O-tetradecanoylphorbol-13-acetate (TPA) on glucose metabolism of the retinal pigmented epithelial (RPE) cells in vitro. Insulin stimulates glucose transport, glucose oxidation and lipogenesis in RPE cells. TPA at low concentrations of insulin increases the rates of glucose transport and glucose oxidation. Depletion of adenosine in RPE cells by adenosine deaminase increases the rate of both glucose transport and 14CO2 formation and improves insulin-sensitivity of both processes. The effects of TPA on RPE cells cannot be explained by the activation of protein kinase C. An alternative possibility is that the effects of TPA on insulin-stimulated glucose disposal in RPE cells is mediated by a change in adenosine concentration and/or the affinity/number of its receptors.

2-Chloroadenosine↗

PubMed1991

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1991

Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells.

In the present study we have demonstrated the presence of calreticulin, a major Ca(2+)-sequestering protein of nonmuscle cells, in a variety of cell types in tissue culture. The protein localizes to the endoplasmic reticulum in most cell types and also to the nuclear envelope or nucleoli-like structures in some cell types. Calreticulin is enriched in the rough endoplasmic reticulum, suggesting a possible involvement in protein synthesis. Calreticulin terminates with the KDEL-COOH sequence, which is likely responsible for its endoplasmic reticulum localization. Unlike some other KDEL proteins, calreticulin expression is neither heat-shock nor Ca(2+)-shock dependent. Using a variety of metabolic inhibitors, we have shown that the pool of calreticulin in L6 cells has a relatively slow turnover and a stable intracellular distribution. In proliferating muscle cells in culture (both L6 and human skeletal muscle) calreticulin is present in the endoplasmic reticulum, and additional intranuclear staining is observed. When fusion of the L6 cells is inhibited with either a high serum concentration or TGF-beta or TPA, the nucleolar staining by anticalreticulin antibodies is diminished, although the presence of calreticulin in the endoplasmic reticulum remains unchanged. In contrast, in differentiated (i.e., fused) muscle cells neither intranuclear nor intracellular staining for calreticulin is present. We conclude, therefore, that calreticulin is abundant in the endoplasmic reticulum in proliferating myoblasts, while it is present in only small amounts in sarcoplasmic reticulum membranes in terminally differentiated myotubes. We propose a model for the domain structure of calreticulin that may explain the differential subcellular distribution of this protein. Because of its widespread distribution in nonmuscle tissues, we postulate that calreticulin is a multifunctional protein that plays an important role in Ca(2+) sequestering and thus that it is the nonmuscle analog of calsequestrin.

Amino Acid Sequence↗

PubMed1991

Adhesion, spreading, and proliferation of cells on protein carpets: effects of stability of a carpet.

In the present report we have investigated the role that the physical properties of substrata play in modulating the effects which components of extracellular matrix (ECM) exert on adhesion, spreading, and growth of retinal pigmented epithelial cells. By simple modifications of conditions for protein adsorption on glass we obtained a set of substrata all coated with proteins of ECM (protein carpets) but with different physical properties. Using these protein carpets we have shown that their stability (desorption rate) in tissue culture conditions varies according to the technique with which they were prepared. Both semiremovable and immobilized carpets are stable, whereas removable protein carpets desorb readily. Therefore, the protein concentration or composition or both may change with time in tissue culture depending on the technique used to prepare the carpet. In addition, efficacy of cell attachment to given protein may vary depending on whether a technique used to prepare the protein carpet involves denaturation of the protein. Adherent cells quickly remove (clear) weakly adsorbed protein carpets and it seems that the carpet removal is a mechanical process. During the carpet removal cells are rounded, which indicates that a spread cell phenotype normally associated with stress fibers and focal contacts occurs when the substratum is rigid enough to sustain cell traction. In addition, substrata lacking the rigidity to support the spread phenotype do not support cell proliferation either.

Animals↗

PubMed1991

Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes".

In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.

Amino Acid Sequence↗

PubMed1991

Characterization of Cobblestone mitral valve interstitial cells.

Mitral valve interstitial cells (MVICs) are important in the structure and function of the valve. In a cell culture system that used explants to harvest MVICs, we found that after several passages, some of the cultures at confluence formed a monolayer of cobblestone-shaped cells (CB type). The rest of the cultures were, however, different, consisting of elongated cells (EL type) that formed overlapping orthogonal patterns. When compared with the MVICs-EL, cells from cultures of MVICs-CB were less adherent to a plastic substratum initially than were MVICs-EL and were more sensitive to trypsinization following culture for 3 to 7 days. Ultrastructurally, the cultures of MVICs-CB showed prominent Golgi's complexes and endoplasmic reticulum and fewer microfilaments when compared with those of MVICs-EL. Most strikingly, the CB cells contained much less alpha-smooth-muscle cell actin compared with that of EL cells. The MVICs-CB were not endothelial cells as they did not show the characteristic dense peripheral band that was present in endothelial cells in a confluent monolayer and showed only trace amounts of fluorescence when incubated with 1,1-dioctadecyl 1-3,3,3,3 tetramethyl-indocarbocyanine-percholate (Dil-)--acetylated low-density lipoprotein compared with large amounts of fluorescence with endothelial cells. Cells that were morphologically similar to MVICs-CB were present in the in vivo valve as well and also formed small islands of cells even in the primary cultures that grew out of the explant. The findings supported the hypothesis that MVICs-CB represent a distinct phenotype of the MVICs that is different from that of the MVICs-EL and is not that of either endothelial or medial smooth-muscle cells.

Actin Cytoskeleton↗

PubMed1990

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata.

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.

Animals↗

PubMed1989

The high-affinity calcium binding protein of sarcoplasmic reticulum. Tissue distribution, and homology with calregulin.

The 55-kDa high-affinity calcium binding protein (HACBP) was first identified and isolated from skeletal muscle sarcoplasmic reticulum (SR). Using polyclonal antibodies raised against the HACBP isolated from skeletal muscle we have identified this protein in cardiac and smooth muscle as well as in non-muscle cells. Although the 55-kDa protein has a size, properties and localization similar to that of calsequestrin, the two proteins are immunologically distinct. The NH2-terminal sequence of uterine HACBP is also completely different from that of calsequestrin but it is identical to that of rabbit liver calregulin, a recently identified calcium binding protein. Indirect immunofluorescence staining of frozen sections and culture cells from a variety of tissues shows that the 55-kDa protein localizes predominantly to junctional SR and T-tubule areas in skeletal muscle, to SR in smooth and cardiac muscle cells, and to ER in a variety of non-muscle cells. These data show that the protein is present in a wide variety of tissues and suggest that it is a protein common for both sarcoplasmic and endoplasmic reticulum membranes.

Animals↗

PubMed1989

Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum.

In this paper I sought to determine how the expression of differentiated traits of chick retinal pigmented epithelial (RPE) cells in vitro can be modulated by varying both the biochemical and the spatial complexity, and the mechanical properties, of the growth substratum. I have used glass derivatized with proteins of a basement membrane extract (nondeformable, two-dimensional substratum) and gels of reconstituted basement membrane extract (viscoelastic, three-dimensional substratum). These two biochemically similar substrata were compared to an inert substratum (untreated glass) and to the native basement membrane of the RPE, i.e., Bruch's Membrane. With immunofluorescence microscopy, I have shown that RPE cells, given space, will spread on their native basement membrane and form stress fibres and focal contacts, analogous to the stress fibres and integrin-, talin-, and vinculin-containing focal contacts of the cells grown on glass. Therefore, the stress fibres and focal contacts present in cultured cells are not artifacts of growth in vitro, but are a natural cellular response to the nondeformability of commonly used tissue culture substrata. The proteins of the basement membrane promote expression of some of the differentiated traits by RPE cells in vitro: however, the fully differentiated phenotype is expressed by RPE cells only when their spreading is prevented by low resilience of a substratum. Basement membrane gels generally are not resilient enough to support RPE cell spreading; however, the cells spread and form stress fibres, and integrin-, talin-, and vinculin-containing focal contacts when they are presented with areas of the gel which locally acquired higher resilience. The extent of cell spreading is determined by the deformability of substratum, hence elastic forces operating within the substratum determine the maximal cell traction allowable and, indirectly, the cytoarchitecture. Therefore, in addition to biochemical composition, the mechanical properties of substrata play important role in regulation of expression of the differentiated phenotype of cells in vitro and, possibly, in vivo.

Animals↗

PubMed1989

Effects of TGF-beta on retinal pigmented epithelium in vitro.

Embryonic chick retinal pigmented epithelium (RPE) has been grown on glass derivatized with covalently bound proteins of basement membrane and treated with transforming growth factor-beta (TGF-beta). In the present paper we show that over the concentration range tested (0.1-10 ng/ml) TGF-beta has no effect on RPE cell proliferation either in the presence or the absence of serum, cell motility and the organization of cytoskeleton-extracellular matrix linkage complexes with respect to their structure and presence of actin, vinculin, talin, integrin and fibronectin. The protein profiles of total cell/ECM extracts of cells grown in the presence or the absence of TGF-beta are similar although some stimulation of protein synthesis and of production of fibronectin-containing extracellular matrix has been detected.

Animals↗

PubMed1989

Cytoskeleton, adhesion, and extracellular matrix of fetal human retinal pigmented epithelial cells in culture.

Small sheets of human retinal pigmented epithelial (RPE) cells isolated from 6- to 9-week-old fetal human eyes were used to establish RPE cells in culture. After 4-5 days in culture the RPE cells in various parts of a colony were examined by immunofluorescence and fluorescence microscopy to determine the distribution of the cytoskeletal proteins tubulin, myosin, F-actin, vimentin, and cytokeratin and the extracellular matrix proteins laminin and type IV collagen. The extracellular matrix components laminin and type IV collagen, which are abundant underneath the RPE cells in the centre of the colony, progressively decrease in amount towards the edge of the colony. Microtubules labeled by antisera to tubulin were present in the cytoplasm of RPE cells throughout the colony. F-actin and myosin were found in stress fibres in the RPE cells at the edge of the colony and in compact circumferential rings circumscribing the apical ends in RPE cells in the centre of the colony. Examination of cell-substratum adhesion patterns by interference reflection microscopy showed that focal contacts were present only in RPE cells at the edge of the colony where they were located at the ends of the stress fibres. The large focal adhesions previously observed in colonies of chick RPE cells, however, were not seen in human RPE cells. Human RPE cells throughout the colony had cytokeratin-containing intermediate filaments as well as vimentin-containing intermediate filaments, whereas only the latter were observed in chick RPE cells. These results indicate that in the general organization of the cytoskeleton and in the distribution of the extracellular matrix components, laminin and type IV collagen, colonies of human RPE cells are similar but not identical to colonies of chick RPE cells.

Actin Cytoskeleton↗

PubMed1988

Adhesion of cells to protein carpets: do cells' feet have to be black?

In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cell-substratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein-covered substrata, their focal contacts may not always appear black in interference reflection microscopy.

Cell Adhesion↗

PubMed1988

Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass.

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of greater than 100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.

Actin Cytoskeleton↗

PubMed1988

Effects of substrata and method of tissue dissociation on adhesion, cytoskeleton, and growth of chick retinal pigmented epithelium in vitro.

In this report we compare attachment, morphology, and growth of retinal pigmented epithelial (RPE) cells isolated by either EDTA or dispase digestion and plated onto either uncoated substrata (plastic or glass) or substrata derivatized by covalent conjugation of proteins of reconstituted basement membrane gel. We show that the derivatized substrata promote better initial attachment and subsequent cell growth than the uncoated substrata. These effects are independent of the method of dissociation of cells from the tissue. Cell morphology, however, is strongly affected by the method used for tissue dispersion. The dispase-dissociated cells are very flat, display a circumferential arrangement of microfilaments and elaborate extensive arrays of vinculin-containing cell-to-cell junctions. In contrast, EDTA-dissociated cells are much less spread, display straight microfilament bundles criss-crossing the cytoplasm and have less extensive cell-to-cell junctions. The protein-derivatized substrata also promote maintenance of differentiated traits, such as pigmentation, by the RPE cells.

Actins↗

PubMed1987

Preliminary characterization of cell surface-extracellular matrix linkage complexes in cultured retinal pigmented epithelial cells.

In this report, we describe the relative distribution of vinculin, talin, and fibronectin in cultured retinal pigmented epithelial cells from chick embryo eyes. We show that in these cells vinculin is present in both focal cell-substratum and cell-cell contacts, whereas talin is present only in the cell-substratum contacts. When cells are double-labeled for talin and fibronectin and viewed at the substratum level, fibronectin is not detectable and talin is concentrated in plaques corresponding to focal contacts. However, when the same cells are viewed at the apical level, both talin and fibronectin are present in a fibrillar pattern. In addition to fibrils which are both talin- and fibronectin-positive, there are areas which are either talin-positive and fibronectin-negative or, vice versa, talin-negative and fibronectin-positive. These observations indicate an interesting variability in the composition of transmembrane linkages in retinal pigmented epithelial cells in vitro.

Animals↗

PubMed1987

Organization of microfilaments in astrocytes that form in the presence of dibutyryl cyclic AMP in cultures, and which are similar to reactive astrocytes in vivo.

This paper deals with changes in the arrangement of microfilaments at various stages during the transformation of astroblasts into reactive astrocytes in the presence of dibutyryl 3',5'-cyclic adenosine monophosphate in vitro. When cultures of two-week-old mouse astroblasts are treated with dibutyryl cyclic AMP, drastic changes occur in cell shape and in the organization of microfilaments, resulting in cells that closely resemble reactive astrocytes in vivo. A thick, prominent ring of microfilaments in such cells which stains strongly with 7-nitrobenz-2 oxa-1,3-diazole-phallacidin, delineates the perikaryon. Electron microscope examination showed that the ring is composed of many smaller bundles of microfilaments running parallel to each other. Prominent bundles of microfilaments radiate from the cell body into the cell processes. Based on the observation of intermediate forms, we propose that the microfilament ring may be important in the development of cell processes in reactive astrocytes.

Actin Cytoskeleton↗

PubMed1986

Spectrin does not redistribute with actin during dBcAMP-induced changes in astrocytes in vitro.

Cells of the astrocyte lineage obtained from mouse neopallium and grown in colony culture have been investigated for a correlation between distributions of F-actin and the common subunit of an erythrocyte actin binding protein, alpha-spectrin (brain fodrin). The cells of the astrocyte lineage at the astroblast stage have F-actin organized in the form of prominent, linearly arranged microfilament bundles. We have demonstrated that spectrin in these cells forms a fine reticulum lining the cell cortex. During the dibutyryl cyclic (dBcAMP)-induced transition from astroblasts to reactive astrocytes, actin-containing microfilaments undergo the dramatic rearrangement from a predominantly linear to a predominantly circumferential spatial organization. remains in the form of a fine reticulum lining the cellular cortex. These remains in the form of a fine reticulum lining the cellular cortex. These findings support the recent notion that spectrin in non-erythroid cells is not essential for maintaining the organization and plasma membrane membrane anchorage of the prominent microfilament bundles.

Actins↗