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M C Alliegro

Publications and source records attributed to M C Alliegro.

At least 19 recordsLinked to original sources
PubMed2001

Pigpen and endothelial cell differentiation.

Endothelial cells can toggle back and forth between differentiated and relatively undifferentiated states with comparative ease. This is an important characteristic, particularly in adult tissues where the constitutive endothelial cell phenotype is quiescent. It enables rapid repair of wounds, renewal of the vascular intima in parts of the circulatory system with high flow and turbulence, and is essential to the cyclic function of reproductive organs. However, the ability to dedifferentiate can be a severe disadvantage when it is subverted to the support of disease processes such as tumor growth and metastasis. The control of endothelial cell differentiation state is, therefore, a matter of significance to investigators of basic developmental mechanism, as well as those studying an array of neovascular disorders. Recently, studies have advanced beyond the identification of extracellular triggers and overt cellular responses to the analysis of signal transduction pathways and nuclear events. This review focuses on the nuclear protein pigpen that is found in the right place at the right time, and with the necessary equipment, to modulate endothelial cell differentiation. We project that when we better understand the relationship of pigpen to its upstream regulators and downstream effectors, we will also have a better understanding of the mechanisms underlying capillary morphogenesis.

Animals↗
PubMed2000

Effects of dithiothreitol on protein activity unrelated to thiol-disulfide exchange: for consideration in the analysis of protein function with Cleland's reagent.

Dithiothreitol (DTT) is widely used to reduce disulfide bonds in the analysis of protein structure and function. However, thiol-disulfide exchange is not the only mechanism whereby DTT can alter protein function. We observe that DTT diminishes the carbohydrate binding activity of a cysteineless mutant of pigpen as well as it inhibits the intact molecule. Lack of inhibition by threitol, a derivative of the four-carbon sugar threose, indicates that the thiol groups of DTT are required for inhibition, and also that DTT is not acting as a simple carbohydrate competitor. Moreover, inhibition of pigpen-carbohydrate binding is not likely due to metal chelation because pigpen binding to carbohydrate is insensitive to EDTA and 1, 10-phenanthroline, which would otherwise be expected to mimic the DTT effect. Our results suggest that DTT can interact with protein domains in the absence of cysteine residues, and that the biochemical reactivity of DTT is not necessarily one and the same with its assumed biochemical specificity.

Amino Acid Sequence↗
PubMed2000

A C-terminal carbohydrate-binding domain in the endothelial cell regulatory protein, pigpen: new function for an EWS family member.

The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [(3)H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.

Animals↗
PubMed1999

Intima-like smooth muscle cells: developmental link between endothelium and media?

The presence of non-contractile smooth muscle cells within the arterial wall raises questions as to their origin and function. These cells abound within the aortae of murine and porcine neonates, but are also present within the intimal and medial layers of adult arteries. They are largely devoid of smooth muscle-associated proteins and manifest an epithelioid form. Their morphological resemblance to endothelial cells prompted us to explore this potential relationship and to investigate their angiogenic properties in three-dimensional collagen gels. Using well-characterized smooth muscle cell lines, displaying either the intima-like (epithelioid) or media-like (spindle-shaped) morphology, we were able to show that intima-like cells share several features in common with endothelial ones and can transform into a media-like phenotype, whereby they irreversibly lose their characteristic pattern of protein expression. Intima-like, but not media-like, vascular smooth muscle cells are capable of forming capillary tubes, and, in co-cultures, can induce media-like ones to participate in this process. Such capillaries consist of a randomly-organized, mixed population of endothelial cells with intima-like or media-like smooth muscle ones. The functional significance of this diversity in smooth muscle cell type is not well understood, but phenotypic plasticity could conceivably figure as an important adaptive response to changes in the local environment.

Animals↗
PubMed1998

Protein heterogeneity in the coiled body compartment.

Coiled bodies are ubiquitous nuclear inclusions of unknown function. Although a considerable list of coiled body components has been assembled in recent years leading to several functional hypotheses, none have yet been borne out by experimentation. Pinpointing coiled body function is difficult in part because each known component molecule has been shown to be present at other sites in the nucleus. Using probes to individual coiled body molecules is therefore likely to yield ambiguous results. From direct observation of coiled body behavior we know that they are dynamic structures, changing in content, size, and number under different physiological conditions. In our studies, we have found that the number of coiled bodies in mammalian endothelial cells is relatively high. Depending on phenotype, quiescent or angiogenic, endothelial cells can average as few as 4 or as many as 15 coiled bodies per nucleus (as opposed to 2 or 3 for most cell types). This can provide certain advantages in the analysis of their dynamics and composition. Moreover, expression of the coiled body protein, pigpen, is sharply regulated as endothelial cells toggle back and forth between the two phenotypes. Using the endothelial cell system, we present several new observations in this report on the dynamics of coiled bodies and their constituent proteins and reinforce prior observations that we consider important but understated in the literature. With antibodies to p80-coilin, pigpen, and fibrillarin, we show that there may be heterogeneity in the coiled body population of individual cells. We demonstrate that the coiled body marker protein p80-coilin can also be found distributed in the nucleoplasm and in apparent association with the nuclear envelope. This suggests that coilin could play a role in some aspect of nucleocytoplasmic exchange. Finally, we correlate the presence of pigpen in a diffuse nucleoplasmic pool with the expression of a phosphatase-sensitive epitope, indicating that subnuclear localization may depend upon the phosphorylation state of the protein. Our results suggest to us that a viewpoint of coiled bodies as part of a fluid trafficking network may be helpful in discerning their cellular functions.

Animals↗
PubMed1997

Novel characteristics of a myosin isolated from mammalian retinal pigment epithelial and endothelial cells.

We have isolated a novel, high Mr protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of approximately 210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of approximately 550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actin-activated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.

Actins↗
PubMed1997

Identification of a new coiled body component.

Coiled bodies are small, round nuclear inclusions that have been identified in many somatic cell types. Equivalent structures are found in the germinal vesicles of amphibian and insect oocytes, known respectively as sphere organelles and Binnenkörper. Their functions are not known, but their molecular composition is being brought to light. In addition to the nucleolar protein, fibrillarin, coiled bodies contain DNA topoisomerase I and an array of RNA processing molecules characteristic of spliceosomes. One coiled body protein absent from nucleoli and spliceosomes, known as p80-coilin, has also been described. We have now identified pigpen, a new member of the EWS family of proteins, as a second protein enriched in coiled bodies. In an earlier report we found that pigpen's structure and expression pattern were suggestive of a role in endothelial cell proliferation and differentiation. In this brief report we characterize pigpen's nuclear compartment and describe its reorganization during mitosis.

Animals↗
PubMed1996

Identification of a new coiled body component.

Coiled bodies are small, round nuclear inclusions that have been identified in many somatic cell types. Equivalent structures are found in the germinal vesicles of amphibian and insect oocytes, known respectively as sphere organelles and Binnenkörper. Their functions are not known, but their molecular composition is being brought to light. In addition to the nucleolar protein, fibrillarin, coiled bodies contain DNA topoisomerase I and an array of RNA processing molecules characteristic of spliceosomes. One coiled body protein absent from nucleoli and spliceosomes, known as p80-coilin, has also been described. We have now identified pigpen, a new member of the EWS family of proteins, as a second protein enriched in coiled bodies. In an earlier report we found that pigpen's structure and expression pattern were suggestive of a role in endothelial cell proliferation and differentiation. In this brief report we characterize pigpen's nuclear compartment and describe its reorganization during mitosis.

Animals↗
PubMed1996

A nuclear protein regulated during the transition from active to quiescent phenotype in cultured endothelial cells.

Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with the FUS gene product, TLS (Translocated in Liposarcoma; Crozat et al, 1993; Rabbits et al., 1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis.

Amino Acid Sequence↗
PubMed1993

Amiloride inhibition of angiogenesis in vitro.

Angiogenesis is important to such processes as normal embryonic development and tissue growth, and is also a central feature of diseases such as diabetic retinopathy and the growth of solid tumors. Understanding the basic events governing angiogenesis has therefore attracted great interest. The ion channel blocking agent, amiloride, has been shown to inhibit angiogenesis in an in vivo model (Lansing et al., '91). This suggested a vital role for Na(+)-coupled transport processes in angiogenesis. A large number of structural analogues of amiloride have been synthesized (Kleyman and Cragoe, '88), and many of these are well characterized with respect to biological activity. These analogues present an opportunity to dissect the process of angiogenesis and identify potentially important physiological events. In this report we describe the effects of amiloride on an in vitro model for angiogenesis employing vascularized tissue explants. Amiloride inhibits capillary morphogenesis completely and reversibly at concentrations as low as 134 microM. It appears to act by blocking endothelial cell proliferation, but not migration. Inhibition is heightened by the introduction of hydrophobic groups on the terminal guanidino nitrogen atom, or on the 5-amino position. An analogue substituted at both of these positions is 30-fold more potent than the parent compound. Of amiloride's known biological activities, these results most closely correlate with the inhibition of Ca2+ transport processes, and thereby suggest an important role for Ca2+ transport in capillary morphogenesis.

Amiloride↗
PubMed1992

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues.

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C8 preparative column with 6% acetonitrile-45% methanol-5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-microns Spin-X cartridges, applied to a reversed-phase C18 column, and eluted in a 0-50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzyl-amiloride.

Administration, Oral↗
PubMed1992

Deployment of extracellular matrix proteins in sea urchin embryogenesis.

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi-laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle.

Animals↗
PubMed1992

On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix.

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.

Animals↗
PubMed1991

Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo.

Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell-echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate.

Age Factors↗
PubMed1991

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.

Animals↗
PubMed1990

In vitro biological activities of echinonectin.

Echinonectin (EN) is a 230-kDa extracellular matrix glycoprotein found in the hyaline layer of sea urchin embryos. Dissociated embryonic cells attached strongly to EN-coated microtiter wells in a centrifugal-based in vitro adhesion assay, suggesting that EN is one of the hyaline layer proteins to which cells adhere in vivo (Alliegro et al., 1988). The present study examines the molecular properties of that adhesion using monoclonal antibodies as probes to block cell attachment, and also demonstrates that EN possesses lectin activity. EN binds tenaciously to agarose-based chromatography resins, such as Sepharose. The sugar-binding activity is associated with the polypeptide component of EN, and not with the carbohydrate moiety. Binding is inhibited with galactose and fucoidan, but not with glucose or locust bean gum. Although functional sites both for polysaccharide binding and for cell attachment are present on each subunit of the EN molecule, the sites appear to be functionally distinct because galactose and fucoidan are completely without effect on cell attachment in vitro. Proteolytic digestion of EN yields a highly limited set of immunoreactive peptides. Digestion with trypsin yields a 20-kDa fragment, chymotrypsin, a doublet at 20 kDa, and 20- and 23-kDa fragments with thermolysin. McAb's directed against these peptides block cell adhesion in vitro, suggesting that they possess the cell attachment domain of EN. This is supported by the observations that trypsin-digested EN is an effective substrate in adhesion assays and that adhesion to the tryptic fragments is also blocked by McAb's to the 20-kDa domain.

Animals↗
PubMed1988

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface.

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.

Animals↗