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Biomedical subjects

M Opas

Publications and source records attributed to M Opas.

At least 37 records · Page 2Linked to original sources
PubMed1998

Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum.

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.

Animals↗
PubMed1998

Direct transdifferentiation in the vertebrate retina.

Transdifferentiation is the process by which differentiated cells alter their identity to become other, distinct cell types. The conversion of neural retina into lens epithelium is one of the most spectacular examples of transdifferentiation. We show that the redirection of cell fate from neural retina to lens and subsequent transdifferentiation is independent of cell replication as it occurs in growth-arrested cell populations. Using DNA ratiometry of individual cells in these cultures we show that, indeed, individual amitotic cells do transdifferentiate. Hence, choice of fate in transdifferentiating cells does not rely on a "community effect" but instead can be categorized as a < > For lack of overt lens progenitors, and most importantly, for its mitotic independence, we conclude that lens colony formation in vitro does occur by direct transdifferentiation and not by clonal proliferation of progenitor cells.

Animals↗
PubMed1997

Calreticulin inhibits glucocorticoid- but not cAMP-sensitive expression of tyrosine aminotransferase gene in cultured McA-RH7777 hepatocytes.

Calreticulin is a ubiquitously expressed Ca2+ binding protein of the endoplasmic reticulum which inhibits DNA binding and transcriptional activation by steroid hormone receptors. In this study the effects of calreticulin on tyrosine aminotransferase (TAT) gene expression in cultured McA-RH7777 hepatocytes was investigated. McA-RH7777 cells were stably transfected with calreticulin expression vector to generate cells overexpressing the protein. The transcriptional activity of the TAT gene, which is glucocorticoid-sensitive and cAMP-dependent, was investigated in the mock transfected McA-RH7777 and in cells overexpressing calreticulin (designated McA-11 and McA-17). In the presence of dexamethasone or the cAMP analog (CTP-cAMP) expression of the TAT gene was induced in mock transfected McA-RH7777 cells by approximately 4.5 and 5 fold, respectively. In McA-11 and McA-17 cells, overexpressing calreticulin, glucocorticoid-sensitive expression of the TAT gene was significantly inhibited, however, the CTP-cAMP-dependent expression of the TAT gene was not affected. The ability of calreticulin to inhibit glucocorticoid-sensitive TAT gene expression but not the cAMP-dependent expression of the gene suggests that the protein affects specifically the action of transcription pathways involving steroid receptors or transcription factors containing KxFF(K/R)R-like motifs. Calreticulin may play an important role in the regulation of glucocorticoid-sensitive pathway of expression of the hepatocytes specific genes during development.

Animals↗
PubMed1997

Heat shock-regulated expression of calreticulin in retinal pigment epithelium.

Calreticulin is a major Ca2+ binding protein in the endoplasmic reticulum of non-muscle cells. In this report we show that calreticulin protein is strongly induced by heat shock. Activation and attenuation of the heat shock transcriptional response is caused by heat shock factor that binds to 5'-flanking sequences of heat shock responsive genes, the heat shock element. The smallest stretch of DNA that shows detectable binding of heat shock factor in vitro contains a two-sequence unit nGAAnnTTCn which exists in the 5'-flanking region of calreticulin DNA (5'-gGAAccCAGcgTTC-3'). The present data provide direct evidence that calreticulin expression can be modulated by heat shock. Thus, our results strengthen the hypothesis that calreticulin, in addition to its function as a cellular Ca2+ store, is a multifunctional protein which performs at least some of its functions from the lumen of the ER.

Animals↗
PubMed1997

Functions of dystrophin and dystrophin associated proteins.

Dystrophin is a protein product of the X-linked gene mutation that is responsible for Duchenne and Becker muscular dystrophies. The protein binds actin and associates with dystrophin-glycoprotein complex to link the cytoskeleton to the extracellular matrix. Defects in the components of the dystrophin-glycoprotein complex are responsible for several phenotypes of muscular dystrophy.

Animals↗
PubMed1996

Endoplasmic reticulum form of calreticulin modulates glucocorticoid-sensitive gene expression.

Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.

3T3 Cells↗
PubMed1996

Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx.

The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.

Adenosine Triphosphate↗
PubMed1996

Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6.

In this report we defined the structural and temporal limits within which calreticulin and calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and calsequestrin are two Ca2+ binding proteins thought to participate in intracellular Ca2+ homeostasis. We show that calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11-12). We also show that the two Ca2+ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of calsequestrin. In the mature rat skeletal muscle, calreticulin and calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulun, but not calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and calsequestrin may be under different control during myogenesis in rat L6 cells in culture.

Animals↗
PubMed1996

Calreticulin modulates cell adhesiveness via regulation of vinculin expression.

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.

Animals↗
PubMed1995

Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.

A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.

Animals↗
PubMed1994

Calreticulin: not just another calcium-binding protein.

In this paper we review some of the rapidly expanding information about calreticulin, a Ca(2+)-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca(2+)-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca2+ storage protein in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

bFGF-induced transdifferentiation of RPE to neuronal progenitors is regulated by the mechanical properties of the substratum.

The N-CAM-negative retinal pigment epithelium (RPE[N-CAM-]) can transdifferentiate into N-CAM-positive neural retina (NR[N-CAM+]) when stimulated by basic fibroblast growth factor (bFGF). In this report we examine whether the properties of the growth substrate affect the fate determination of the presumptive RPE. We used a system of biochemically related substrata with different mechanical properties, that is hydrated basement membrane gels (BM gels) and carpets of immobilized basement membrane proteins (BM carpets). We examined the effects of bFGF on RPE grown on either BM gels or BM carpets and compared them with the effects of bFGF on RPE cells grown on their native basement membrane. We show that bFGF provides the stimulus necessary to redirect the choice of fate of the presumptive RPE[N-CAM-] from the RPE pathway into the neural pathway. However, the mechanical properties of the substratum determine the extent to which a neural phenotype is expressed by the transdifferentiating cells. RPE[N-CAM-] transdifferentiates into a pleomorphic neuroepithelium[N-CAM+] on rigid, two-dimensional BM carpets, into a pseudostratified neuroepithelium[N-CAM+] on highly malleable BM gels, and into a stratified, NR[N-CAM+]-like neuroepithelium on its native basement membrane, which is of intermediate rigidity. The newly formed NR, except for the inverted polarity, has a morphology corresponding to, and expresses markers in a distribution appropriate for, the equivalent stage of retinal histodifferentiation in the embryo. We also show that bFGF is not a mitogen for the presumptive RPE cells, while it is a potent one for the presumptive NR.

Animals↗
PubMed1994

Generation and characterization of antibodies to adhesion-related molecules of retinal pigment epithelial cells.

We have generated antisera to pig retinal pigment epithelial (RPE) cells and to their fractionated membrane proteins. The antisera have been screened functionally by adhesion inhibition assays, and morphologically with immunofluorescence microscopy of cultured cells and frozen sections of the retina. From these sera, we have affinity purified five monospecific antibodies to the RPE surface molecules having molecular weights of 220, 180, 110, 85 and 70 kDa, which effectively inhibit cell-substratum attachment. The 220 and 85 kDa antigens are localized to focal contacts of the cultured cells and are seen in patches in the intact RPE layer in frozen sections of eye tissues. Immunofluorescence microscopy shows that the 180, 110 and 70 kDa antigens localize predominantly to cell-cell junctions. Hence, these antigens may participate in both cell-cell and cell-substratum adhesion in RPE cells. Staining patterns obtained with confocal microscopy of frozen sections of the retina demonstrate that these antibodies are RPE-specific. These adhesion-related molecules may play a role in maintenance of the morphological and functional integrity of RPE.

Animals↗
PubMed1994

Major retinal cell components recognized by onchocerciasis sera are associated with the cell surface and nucleoli.

PURPOSE: Cellular localization of the components recognized by onchocerciasis autoantibodies has not been investigated in any detail in cultured retinal cells. This study sought to examine, in cultured retinal cells, the subcellular localization of major components that cross-react with onchocerciasis sera. METHODS: Immunofluorescence confocal laser scanning microscopy and Western blot analysis were carried out on adult pig retinal cells. RESULTS: The onchocerciasis sera contain antibodies cross-reacting strongly with components of the surface and nucleoli in both the cultured retinal pigment epithelial and neural retinal cells. These epitopes are not recognized by the control sera obtained from noninfected individuals residing in an onchocerciasis hyperendemic area, and from those with or without ocular disease who have never been in any of the onchocerciasis hyperendemic countries. Double-labeling immunofluorescence microscopy does not detect any colocalization of a putative onchocerciasis autoantigen, calreticulin, and those cellular components recognized by onchocerciasis sera in either cell type. Furthermore, none of the onchocerciasis sera tested recognized recombinant calreticulin by Western blot analysis. CONCLUSIONS: Major epitopes for onchocerciasis anti-retinal autoantibodies are associated with the surface and nucleolus components of retinal cells. Interaction of the onchocerciasis antibodies with the retinal cell surface molecules may play an important role in the development of ocular diseases initiated by the damage of retinal cells. Furthermore, the finding that the cellular components recognized by onchocerciasis sera do not colocalize with calreticulin, taken together with the observation of lack of recognition of recombinant calreticulin by these sera on Western blots, suggests that calreticulin is not a major onchocerciasis autoantigen.

Animals↗
PubMed1994

Involvement of non-receptor protein tyrosine kinases in expression of differentiated phenotype by cells of retinal origin.

Regulation of phenotypic expression in epithelia in general, and of two epithelia of the retina, the neural retina and retinal pigment epithelium in particular, is dependent on interactions with extracellular environment. Extracellular environment may comprise acellular substrata as well as other cells. Non-receptor protein tyrosine kinases are involved in transmembrane transmission of signals from extracellular milieu, via the cytoskeleton to the nucleus. We describe distribution of these kinases in cells of retinal origin and show that two of them, pp125FAK and pp60c-src redistribute intracellularly in a differentiation-dependent manner. Next we discuss roles that adhesion-related non-receptor protein tyrosine kinases might play in phenotypic expression by the retinal epithelia.

Animals↗
PubMed1993

Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner.

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor-mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), affects RPE cell adhesion in a substratum-dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long-lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long-term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell-cell contacts, causing an alteration in the shape ("squaring") of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long-term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long-term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Calreticulin.

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Amino Acid Sequence↗