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PubMed1991

Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.

PECAM-1 is a 130-120-kD integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. Previous studies have shown that it is a member of the immunoglobulin gene superfamily and that antibodies against the bovine form of this protein (endoCAM) can inhibit endothelial cell-cell interactions. These data suggest that PECAM-1 may function as a vascular cell adhesion molecule. The function of this molecule has been further evaluated by transfecting cells with a full-length PECAM-1 cDNA. Transfected COS-7, mouse 3T3 and L cells expressed a 130-120-kD glycoprotein on their cell surface that reacted with anti-PECAM-1 polyclonal and monoclonal antibodies. COS-7 and 3T3 cell transfectants formed cell-cell junctions that were highly enriched in PECAM-1, reminiscent of its distribution at endothelial cell-cell borders. In contrast, this protein remained diffusely distributed within the plasma membrane of PECAM-1 transfected cells that were in contact with mock transfectants. Mouse L cells stably transfected with PECAM-1 demonstrated calcium-dependent aggregation that was inhibited by anti-PECAM antibodies. These results demonstrate that PECAM-1 mediates cell-cell adhesion and support the idea that it may be involved in some of the interactive events taking place during thrombosis, wound healing, and angiogenesis.

Amino Acid Sequence↗
PubMed2001

Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro.

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.

Bacterial Adhesion↗
PubMed1984

Chick embryonic pigmented retina is one of the group of epithelioid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin.

Using sodium dodecyl sulphate/polyacrylamide gels to analyse detergent-insoluble residues, and indirect immunofluorescence, we have found that the major protein of intermediate filaments in cultures and freshly explanted fragments of chick embryonic retinal pigment epithelium (RPE) is vimentin. Moreover, these cells also fail to stain with antibodies against cytokeratins and most components of true desmosomes (maculae adhaerentes). Staining with anti-vinculin antibody suggests that the principal intercellular junction is the zonula adherens. Thus although RPE is an epithelium according to all other criteria, it belongs to a group of tissues (including vascular endothelium, iris and lens-forming epithelium) that have intermediate filaments composed of vimentin and possess neither cytokeratins nor desmosomes. That a tissue can be fully epithelial by other criteria, whilst lacking these components, is in agreement with other work, which has shown a lack of effect of micro-injection of antibodies to cytokeratin, and of the suppression of desmosome formation, on epithelial organization in culture. Although our observations were made solely on chick embryonic tissue, we suggest that published ultrastructural studies are consistent with the possibility that RPE of other species, including human, may lack true desmosomes.

Animals↗
PubMed2004

Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.

Interstitial collagen I stimulates microvascular endothelial cells to form solid cords that imitate precapillary structures found during angiogenesis. Time-lapse microscopy identified cell retraction and disruption of cell-cell contacts as early critical steps in collagen I-induced capillary morphogenesis. These early stages paralleled collagen I activation of Src kinase and GTPase Rho through beta1 integrins. The Src inhibitor PP2, dominant-negative Src, and Rho inhibitor exoenzyme C3 transferase each inhibited collagen I induction of actin stress fibers that mediate cell retraction and each inhibited capillary morphogenesis. Collagen I also disrupted VE-cadherin from intercellular junctions through a Src-dependent mechanism; both the Src inhibitor PP2 and dominant-negative Src preserved VE-cadherin localization to regions of cell-cell contact. An active Src mutant disrupted VE-cadherin and cell-cell contacts similarly to collagen I. In sharp contrast, laminin-1 did not induce capillary morphogenesis, and laminin-1 did not induce activation of Src or Rho. Rather, laminin-1 induced persistent activation of the GTPase Rac. Thus, these studies identify activation of Src and Rho as key mechanisms by which collagen I provokes capillary morphogenesis of microvascular endothelial cells, and they define marked differences between the functions of collagen I and laminin-1 in regulating endothelial cell morphogenesis.

ADP Ribose Transferases↗
PubMed1997

Cytotoxicity of a low molecular weight fraction from Aloe vera (Aloe barbadensis Miller) gel.

The cytotoxicity of a low mol. wt fraction (LMWF) obtained from Aloe vera gel was determined by two different assays. Firstly, exposure of monolayers of chicken fibroblasts to LMWF induced disruption of intercellular junctions and detachment of individual cells from the bottom of the flask, with formation of cell-free gaps in the monolayer. Secondly, LMWF inhibited the production of reactive oxygen species by human polymorphonuclear leukocytes stimulated by zymosan, as followed by luminol-dependent chemiluminescence. The toxic activity of LMWF was compared to that of sodium dodecyl sulfate (a well-known toxic substance), aloe-emodin and aloin (an anthraquinone and its precursor present in Aloe vera cortex) using the chemilumescence assay, and was found to be of similar potency to these toxic substances on a weight-to-weight basis. These results confirm that Aloe vera gel contains toxic low mol. wt compounds, and every effort must be made to limit the amount of these toxins in the commercially prepared Aloe vera gel products.

Aloe↗
PubMed1978

Ultrastructure of effector--target cell interaction in secondary cell-mediated lympholysis.

The ultrastructure of the secondary cell-mediated lympholysis (CML) reaction and the effects on interacting lymphocytes of colchicine, cytochalasin B, and effector cell-specific antisera were examined using transmission and scanning electron microscopy. Surface labelling of cytotoxic secondary effector cells with cationized ferritin allowed them to be distinguished from unlabelled target lymphocytes. Effector--target interactions were characterized by intercellular junctions involving extensive areas of membrane apposition and interdigitation and extension of pseudopod-like processes by the effector cell. The abolition of such interactions when effector populations were pretreated with anti-Ly2 sera plus complement demonstrated target cell destruction in secondary CML to be dependent on the activity of restimulated cytotoxic T lymphocytes. Cytochalasin B and colchicine dramatically decreased the numbers of specific effector--target cell interactions observed. Although the data presented do not allow the possible activity of soluble lytic factors associated with the effector cell surface to be ruled out, they suggest that target cell lysis in the secondary CML system examined results from immune-specific binding of alloantigen-sensitized effectors to targets and osmotic effects which follow localized disruption of the target cell membrane.

Animals↗
PubMed1981

Manipulation of cell-cell and cell-substratum interactions in mouse mammary tumor epithelial cells using broad spectrum antisera.

Two antisera were raised in goats against material shed by two different mammary epithelial cell lines into serum-free culture medium. These antisera, when added to the medium of intact, growing mouse mammary tumor cells in the absence of complement, cause distinct and dramatic alterations in cell morphology and adhesiveness. One antiserum (anti-SFM I) causes mouse mammary tumor epithelial cells to round and detach from the substratum. Treatment with the other antiserum (anti-SFM II) does not affect cell-substratum interactions, but causes the cells to convert from an epitheloid to a fibroblastic morphology. Statistical analysis of transmission electron micrographs of control and antibody-treated cells indicates that treatment with anti-SFM II is associated with a substantial reduction in the extent of intercellular junctions, particularly desmosomes. To identify the components with which the two antisera interact, nonionic detergent extracts of mouse mammary tumor cells were fractionated, and the ability of various fractions to block the morphological effects of either antiserum was determined. The whole Nonidet P40 (NP40) extract of the epithelial cells blocked the effects of both antisera. After the extract was subjected to ion exchange and lectin affinity chromatography, two separate fractions were obtained. One fraction blocks and anti-SFM I induced rounding and detachment of cells from the substratum. The second fraction blocks the effects of both antisera. The isolation of the former fraction, which has highly restricted number of components, represents a significant first step toward identifying the surface membrane molecule(s) involved in cell-substratum adhesion in epithelial cells.

Animals↗
PubMed2002

Distinctive alterations of invasiveness, drug resistance and cell-cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant.

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (PA) secretion. MCF-7 cells did not show any tendency to invade, whatever the mode of culture. These results show that 3D-cultures as spheroids distinctively altered structural features of parental and MDR-MCF-7 cells. In MCF-7 cells, 3D-culture increased cell-cell contacts and drug resistance; in MDR-MCF-7 cells, it induced invasive properties.

Adenocarcinoma↗
PubMed1987

Postnatal development of the ovarian bursa of the golden hamster (Mesocricetus auratus): its complete closure and morphogenesis of lymphatic stomata.

The golden hamster ovarian bursa was studied by light and electron microscopy to clarify the process of its complete closure and the development of lymphatics that leads to morphogenesis of stomata. The results were as follows. 1) The bursa completely closed at 9 days of age primarily due to development of the mesotubarium superius. 2) With the closure, the ovary and bursa became closely apposed, and most of the original bursal cavity disappeared. 3) Between 9 and 12 days of age U-shaped folds of the bursal mesothelium began to invade the connective tissue of the bursa. 4) Widening of the internal angle of the U-shaped folds contributed to reappearance of the bursal cavity, and thus separation of the bursa from the ovary. It also contributed to future geometrical proximity of lymphatics to the cavity of the bursa. 5) The separation of the bursa from the ovary began as early as 12 days of age in the cephalic half of the bursa. It occurred remarkably late in the caudal half. Juxtaposition of the window portion of the bursa to the ovary remained in some adult animals. 6) Development of lymphatics in the cephalic half of the bursa was divided into two stages, before and after days 21-24 of life. In the first stage, lymphatics grew in the submesothelial connective tissue, and the framework of lymphatics was formed. In the second stage, lymphatics extended small branches to form the submesothelial plexus or lymphatic lacuna. 7) Intercellular junctions between contiguous lymphatic endothelial cells were mostly tight and desmosomelike. Open junctions were, if they occurred at all, rare. (8) A smooth-surfaced area lined with the lymphatic endothelium was found in the bursa on day 27 of life, before the initiation of ovulation. Valvelike stomal orifices were absent before the initiation of ovulation and extremely rare even after the first ovulation. They were commonly present in the bursae after the fourth ovulation, however. The process of complete closure of the ovarian bursa is very complex and may be related to the later development of the bursal mesothelium and lymphatics. Some liplike stomal orifices are of purely developmental origin. However, all valvelike stomal orifices are assumed to be formed as a result of damage to the bursal mesothelium, as well as to the submesothelial connective tissue and lymphatics, by repetition of ovulation. It is possible that liplike stomal orifices may be formed in the process of repairing the damage.

Age Factors↗
PubMed1997

Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro.

The composition of fluids within the bovine oviduct and uterine lumen, important in fertilisation and early embryonic development, is ultimately determined by the transport properties of the epithelial cells which line the lumen. A preparation has therefore been devised to study the role of these cells in oviduct and uterine fluid formation. Pure preparations of epithelial cells, as judged immunocytochemically, were isolated by enzyme digestion, and grown on collagen filters in primary culture. The cells re-establish intercellular junctions to form a confluent epithelial layer. Serial samples from the apical and basal media were analysed for K+, Na+, Ca2+, glucose and lactate. Bovine oviduct epithelial cells maintained gradients of K+ and Ca2+ (apical > basal) for up to 14 days after confluence, while bovine uterine epithelial cells maintained apical > basal gradients of K+. Both types of epithelium exhibited a small transepithelial electrical potential difference and a higher uptake of glucose and production of lactate in the basal, as opposed to apical medium. There were no consistent differences in any of these parameters with the stage of the oestrous cycle at which the cells were removed. The data indicate that bovine oviduct and uterine epithelia may be isolated and grown as polarised layers in primary culture. The preparations will now enable the mechanisms underlying the secretion of ions and non-electrolytes to be determined.

Animals↗
PubMed1999

Pathogenesis of African horse sickness: ultrastructural study of the capillaries in experimental infection.

African horse sickness (AHS) was induced in five horses by inoculation, to determine the ultrastructural changes in endothelial cells of capillaries in the myocardium, lung, spleen and liver. The animals developed cardiac and mixed forms of the disease. Alterations detected in the endothelial cells of the vessels of infected animals included: the presence of structures associated with viral infection, hypertrophy, degenerative changes, appearance of cytoplasmic projections, changes in permeability, alteration of intercellular junctions, loss of endothelium, subendothelial deposition of cell debris and fibrin, and vascular repair. In association with these changes, oedema, haemorrhages and microthromboses were detected, particularly in the myocardium and lung. This study showed that infection of, and changes to, the capillary endothelial cells of the organs under study was independent of the form in which the disease manifested itself but was dependent on the organ and blood vessel type. Thus, different levels of viral tropism were observed for the endothelial cells of the vessels in different organs. Viral infection was commonest in the endothelial cells of myocardial vessels, followed by those in the lung, whereas in the spleen and liver, endothelial cell infection was rare and, in the case of the liver, limited to the interstitial capillaries.

African Horse Sickness↗
PubMed2005

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components.

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.

14-3-3 Proteins↗
PubMed1986

Ultrastructural quantitative characterisation of sinus and atrioventricular nodal cells in the developing caprine heart compared with ordinary atrial and ventricular myocardial cells.

Cellular growth and changes in subcellular components as well as in intercellular junction structure were investigated quantitatively in sinus and atrioventricular nodes in comparison with right atrial and left ventricular myocardial cells of the goat heart in fetuses aged 6, 8, 10, 12, 14, 16 and 18 gestational weeks, young animals aged 1 and 3 postnatal weeks, and adults. At the earliest stage examined (sixth gestational week) no clear difference was recognised, in the patterns of star diagrams representing cellular characteristics, between nodal cells and ordinary cardiac muscle cells, except for a larger number of myofibrils in the ordinary cells. Atrial specific granules also appeared in the atrial cells at this stage. In general, initial signs of differentiation in ordinary cardiac muscle cells were detected at the sixteenth gestational week when transverse tubule formation occurred in the ventricular muscle cells. Throughout the developmental period, no appreciable alterations in the ultrastructural features were found in the sinus nodal cells whereas the atrioventricular nodal cells revealed slight changes at the later fetal and adult stages.

Animals↗
PubMed2004

Optisol vs Dexsol as storage media for preservation of human corneal epithelium.

PURPOSE: To compare the efficacy of two storage media, Optisol GS and Dexsol, in preservation of donor corneal epithelium. METHODS: A total of 12 pairs of corneas not suitable for transplantation, all with intact epithelium, were used in this study, with one cornea of the pair stored in Optisol GS and its other counterpart in Dexsol. At each of three durations of storage--1, 2, and 4 days--four of these paired corneas were prepared for light microscopy and scanning and transmission electron microscopy. Another four pairs of control cornea were prepared in the same way and placed in universal fixative. MAIN OUTCOME MEASURES: Evaluation of the corneas was made by two observers masked as to the identity of the storage medium and length of storage. Loss of epithelial cells was evaluated by light microscopy. The attachment of the epithelium to the basement membrane,cellular integrity, intercellular junctions, and intracellular organelles were evaluated and compared by electron microscopy. RESULTS: The magnitude of epithelial loss correlated with the length of storage time. Control corneas maintained normal epithelium with preservation of all epithelial cell layers. Corneas stored for 1 day had minimal damage of the epithelium. Corneas stored for 2 days had a slight increase in epithelial damage, and corneas stored for up to 4 days showed a marked increase in epithelial damage. There were no significant differences between the two storage media. The basal cell layer was maintained in both the media at all time points, usually in good condition with mild-to-moderate damage in some cases. CONCLUSIONS: Loss of donor epithelium is related mainly to the length of storage and is similar in both Optisol GS and Dexsol. The storage time should be less than 4 days,especially when performing penetrating keratoplasty on patients with ocular surface disorders.

Aged↗
PubMed1980

Blood-brain barrier: a definition of normal and altered function.

The anatomical component of the blood-brain barrier (BBB) has been shown to be the cerebral capillary. These capillary endothelial cells are connected by continuous tight intercellular junctions and under normal conditions do not demonstrate transendothelial channels or pinocytotic vesicles. The rate that substances penetrate the BBB is related to molecular size, lipid solubility, and the presence of a specific carrier-mediated transport system. This latter mechanism for transendothelial passage is utilized for the movement of a wide variety of biologically important compounds such as sugars, amino acids, and organic acids. In certain pathological conditions, the permeability of the BBB is altered so that normally excluded plasma proteins and fluid enter the brain extracellular space, with the subsequent development of cerebral edema. In other abnormal conditions, alterations in the specialized transport systems operating across the cerebral capillary result in adverse changes in cerebral and neurotransmitter metabolism. An understanding of the unique properties of the BBB and of the changes that occur in pathological conditions has allowed the development of rational therapeutic strategies for a wide variety of diseases of the central nervous system.

Aging↗
PubMed1983

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates.

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.

Animals↗
PubMed2006

Calcium-independent desmosomes of keratinocytes are hyper-adhesive.

Desmosomes in tissues are resistant to disruption by chelation of extracellular calcium. It has been suggested that this represents a hyper-adhesive state of these intercellular junctions that is crucial for the maintenance of epidermal integrity. Desmosomes change to a lower affinity, calcium-dependent adhesive state when cells are cultured at low density or when an intact epithelial cell sheet is wounded. Here we demonstrate that cells of the immortalized human keratinocyte line HaCaT acquire calcium-independent desmosomes in confluent culture. An adhesion assay shows that HaCaT cells with calcium-independent desmosomes are more cohesive than cells with calcium-dependent desmosomes. This assay relates directly to desmosomal adhesion because it involves splitting of the desmosomal adhesive material. Moreover, the difference in adhesiveness between calcium-dependent and calcium-independent desmosomes involves no quantitative change in the known protein composition of desmosomes. Instead, switching between the two adhesive states can be achieved by activation or inhibition of protein kinase C (PKC), suggesting a direct effect of PKC signalling on desmosomal adhesion. These results provide direct support for the concept of hyper-adhesiveness in desmosomes.

Calcium↗
PubMed2002

Clustering of MHC-peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains.

Protein reorganization at the interface of a T cell and an antigen-presenting cell (APC) plays an important role in T cell activation. Imaging techniques reveal that reorganization of particular receptor-ligand pairs gives rise to an intercellular junction, termed the immunological synapse. In this synapse antigenic peptides associated with major histocompatibility complex (MHC) molecules form multimolecular arrays on the APC side, engaging an equivalent number of clustered T cell receptors (TCRs) on the T cell. The accumulation of MHC molecules carrying cognate peptide in the APC-T cell interface was thought to depend on the specificity and presence of TCRs. Recent evidence, however, suggests that the APC is equipped to preorganize MHC-peptide complexes in the absence of T cells. To this end, MHC molecules become incorporated into two types of membrane microdomains: (i) cholesterol- and glycosphingolipid-enriched domains, denoted lipid rafts, that preconcentrate MHC class II molecules; and (ii) microdomains made up of tetraspan proteins, such as CD9, CD63, CD81 or CD82, that mediate enrichment of MHC class II molecules loaded with a select set of peptides. It follows that the integrity, composition and dynamics of these microdomains are candidate determinants favoring activation or silencing of T cells.

Animals↗