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PubMed1983

Spatial distribution of proteins specific for desmosomes and adhaerens junctions in epithelial cells demonstrated by double immunofluorescence microscopy.

The spatial relationships between the protein constituents to two junctional structures, adhaerens junctions and desmosomes, were determined by double immunofluorescence microscopy using marker proteins specific for these structures. Adhaerens junctions were visualized by immunofluorescent labeling for the membrane-associated protein vinculin and by their association with actin filaments. Desmosomal components were identified by labeling with antibodies to a group of minor desmosomal plaque proteins (DP1 antigens) and their association with filaments stained by cytokeratin antibodies. Double immunofluorescence microscopy of these components was performed in several tissues and cultured cells, including intact intestine, dissociated intestinal cells, and two morphologically different types of epithelial cells, cultured bovine kidney (MDBK), and mammary gland (BMGE) epithelial cells. This allowed the direct demonstration that each filament system is associated exclusively with its specific membrane-bound junctional protein. Vinculin and DP1-protein were found in distinct sites in the subapical intercellular junctional complex of intestinal epithelium and MDBK cells. Cell-substrate focal contacts contained vinculin and actin and showed no apparent relationships to the tonofilament system whereas intercellular contacts of BMGE cells were characterized by positive staining for DP1-protein and associated cytokeratin filaments. Immunolabeling of the cultured cells at different intervals after plating for the cytoskeletal elements and their membrane anchorage proteins was used to determine the temporal sequence of their organization. We propose that this approach may be used for the molecular definition and identification of cellular contacts and junctions as well as for studies of junction topology, dynamics and junction-cytoskeleton interactions, and junction biogenesis.

Actins↗
PubMed1982

"Hemogenic endothelium" of the embryonic aorta: Does it exist?

Aggregates of intravascular, presumably endothelial-derived, undifferentiated cell were found in the aorta of 14-15 day Mongolian gerbil embryos, in several 10 mm pig embryos, in one 10 day mouse embryo and in one 9.5 mm human embryo. As in other species in which they have been identified, these "aortic cell clusters" generally occurred in groups of 25-100 cells and were typically observed adherent to the ventral luminal wall of the abdominal aorta during a brief period in gestation. The present electron microscopic study shows that these cells possess many characteristics in common with undifferentiated primitive blood cells of the yolk sac blood island. Their in situ origin is supported by ultrastructural similarity to underlying endothelium as well as the presence of numerous intercellular junctions among themselves and with subjacent endothelium. It is suggested that they may be morphologically undifferentiated hemopoietic precursor cells. Hypotheses are proposed for the mechanism of their origin from aortic endothelium.

Animals↗
PubMed1986

Immunohistochemical and ultrastructural study of Sertoli cells in androgen insensitivity.

The ultrastructure of a perfusion-fixed gonad in a case of androgen insensitivity was studied using thin sections and freeze-fracture replicas. The distribution and arrangement of intermediate filaments in Sertoli cells was visualized immunohistochemically using an antibody against vimentin. Leydig cells lacked Reinke crystals, but contained all of the cytoplasmic organelles involved in steroid synthesis and additionally several lysosomes. The basement membrane and the basal lamina of the testicular tubules were considerably thickened. The testicular tubules consisted of gonocytes and Sertoli cells which had an immature nuclear structure, incomplete development of intercellular junctions and a primitive distribution pattern of intermediate cytoplasmic filaments. The previously reported differences in electron density of Sertoli cell cytoplasm are a non-specific feature without significance to Sertoli cell maturation.

Adult↗
PubMed1994

Molecular aspects of the loss of cell adhesion and gain of invasiveness in carcinomas.

It has been realized that the loss of epithelial differentiation in carcinomas, which is accompanied by higher mobility and invasiveness of the tumor cells, is often a consequence of reduced intercellular adhesion. A variety of recent reports have indicated that the primary cause for the 'scattering' of the cells in invasive carcinomas is a disturbance of the integrity of intercellular junctions. It has also been suggested that during invasion, carcinoma cells convert to a sort of mesenchymal stage, as do normal epithelial cells during development. This paper discusses permanent and transient molecular mechanisms which lead to the impairment of junction integrity of epithelial cells and thus to the progression of carcinomas towards a more invasive state.

Animals↗
PubMed1996

Structural recovery of small arteries following clamp injury: a light and electron microscopic investigation in the rat.

Endothelial injuries were induced in the left common iliac arteries (1 mm in diameter) of rats, by the placement of 1 mm Scovell-Lewis microvascular clamps for 5 minutes, to create a lesion in which to quantitate the rate and degree of cellular regeneration. The left (clamp-injured) and right (control) iliac arteries from the 15 rats used in this study were viewed with the electron microscope at 2, 7, and 14 days after clamping, and the clamp sites were analysed morphometrically. At 2 days there was only minimal denudation of the endothelium; most cells were disoriented and showed some signs of traumatic injury. By 7 days there was a completely continuous endothelial lining, but there was also evidence of increased cytoplasmic activity in these cells, as well as a statistically significant simplification in their intercellular junctional morphology. These changes persisted at 14 days after injury, but they were less pronounced. Smooth muscle cells in the media were relatively unaffected by the trauma in the first 2 days after clamping. However, they exhibited a change of phenotype from contractile to synthetic by 7 days after injury. By 14 days most smooth muscle cells had reverted back to the contractile phenotype, with little evidence of residual damage. These studies reveal that the reconstitution and regeneration of the endothelium is very rapid following clamp injury, but that significant residual ultrastructural changes in the interendothelial junctions persist for at least 14 days after injury. These findings indicate that there is potential for subsequent pathological changes in sites of vascular clamp injury.

Animals↗
PubMed1992

The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes.

The distribution of adherens junction (AJ) components was investigated in cultured heart myocytes. These cells, derived from either newborn rats or chick embryos, develop elaborate arrays of myofibrils which become extensive and laterally aligned following several days in culture. The Z-disks in these cells, visualized by immunolabeling with antibodies to muscle-specific alpha-actinin, exhibit a characteristic periodicity of about 2 microns and are in register with those of neighboring myofibrils throughout the sarcoplasm. Vinculin, in these cells, associates with intercellular AJ and cell-matrix adhesions. In addition, this protein is detected in periodic bands located along the lateral cell membranes corresponding to "costamers" previously described by Pardo, J.V., Siliciano, J.D. and Craig, S.W. (Proc. Natn. Acad. Sci. USA, 80, 1008). Similarly, N-cadherin, which is predominantly associated with intercellular junctions, is also detected in periodic striations located mainly on the dorsal and lateral cell surfaces. Using computer-aided three-dimensional microscopy confirmed that these vinculin- and N-cadherin-containing structures are located in extrajunctional sites, apparently associated with Z-disks of peripheral myofibrils. Based on these findings an alternative pathway is proposed for the assembly of vinculin and N-cadherin, which is not triggered by adhesive interactions with extracellular surfaces but rather by interactions at the membrane-cytoplasm interphase with the periphery of the pre-assembled myofibrils. Moreover, we present evidence that antibodies to N-cadherin, which are capable of blocking AJ formation in culture, have an inhibitory effect also on the development and alignment of myofibrils. We discuss the functional significance of the "costameric" organization of vinculin and N-cadherin and consider its involvement both in the lateral alignment of neighboring muscle cells and in the stabilization of developing myofibrils.

Actins↗
PubMed1992

Chemopreventive effects of green tea components on hepatic carcinogenesis.

Catechin components of green tea have been shown to possess anticarcinogenic properties possible related to their antioxidant activity. In the present study, a catechin containing green tea extract (GTE) was examined for its effect on three previously defined properties of liver tumor promoters, induction of cytolethality, inhibition of gap junctional intercellular communication, and induction of cell proliferation. Hepatocytes from male B6C3F1 mice were isolated and placed in primary culture. The effects of GTE of oxygen free radical-induced cytolethality was examined by coincubating GTE with the oxygen radical generating compounds paraquat, glucose oxidase (GO), and xanthine oxidase (XO). GTE prevented the induction of hepatocyte cytolethality by GO, XO, and paraquat in a dose-responsive manner. Similarly, GTE prevented the inhibition of gap junctional-mediated intercellular communication (measured by lucifer yellow dye coupling) by phenobarbital, lindane, and paraquat in a dose-dependent manner. The effect of GTE on hepatocyte DNA synthesis was examined in male mice containing preneoplastic liver lesions induced by diethylnitrosamine. GTE significantly decreased the labeling index in hepatic preneoplastic foci from animals treated with phenobarbital for 7 days. These studies suggest that the previous reported anticarcinogenic activity of green tea may be related to its effect on the tumor promotion stage of the cancer process.

Animals↗
PubMed1991

A common epitope is shared by ciliary rootlets and cell-cell adherens junctions in ciliated ependymal cells.

Using immunoblot, light and electron immunocytochemistry, we investigated the presence and the localization of polypeptides cross-reacting with the monoclonal antibody CC.310 (mAb CC.310), which is mainly directed against a 175K (K = 10(3) Mr) ciliary rootlet protein. In hypothalamic ependymal cultures, the unique antigen recognized by mAb CC.310 was associated with the Triton X-100-insoluble fraction in these cultures and electrophoretically migrated to these cultures and electrophoretically migrated to 94K. mAb CC.310, which appears to be a very suitable marker for ciliated ependymocytes, allowed us to observe ciliogenesis during the growth of the ependymal cultures, from a single spot in each undifferentiated ependymal cell to a massive labeling in ciliated ependymal cells. In fully differentiated ciliated ependymocytes, mAb CC.310 strongly reacted with fibrous structures corresponding to ciliary rootlets, as confirmed by ultrastructural observations. In addition, a weaker immunostaining was also found along the intercellular junctions, and showed that proteins sharing a common epitope are located in ependymal ciliary rootlets and near adherens-type junctional complexes. Immunofluorescence studies confirmed the presence of positive labeling at the level of junctional complexes between cells in two epithelial lines, HeLa and PtK2, in which mAb CC.310 mainly reacted with one polypeptide of 85K.

Animals↗
PubMed1981

The fine structure of snake myocardium.

Snake myocardium has been studied using standard methods and tannic acid mordanting. In both atria and ventricle, cells were arranged in fascicles with little connective tissue, up to 20 cells per fascicle, and few differences between cells of atria and ventricle. Cell (fiber) size varied from 8 to 12 micrometers in the nuclear area with a few cells up to 14 micrometers in ventricle, and cells generally were spindle-shaped, tapering toward the extremities where relatively simple intercalated discs were seen. Few myofibrils per cell were present and poorly delineated. Transverse tubules were absent and sarcoplasmic reticulum was poorly developed and, apparently, even absent from some cells. All cells contained micropinocytotic vesicles associated with the surface plasmalemma and subsarcolemmal caveolae. In addition to intercalated discs, intercellular junctions showed spot desmosomes, small and sparse gap junctions and fasciae adherentes of two types, one associated with Z-bands, one independent of Z-bands. Nerve fibers and nerve endings were numerous, particularly in atrium. These findings are discussed in relation to the structure of mammalian myocardium.

Animals↗
PubMed1979

The terminal web. A reevaluation of its structure and function.

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.

Animals↗
PubMed1979

Electrical properties of an excitable epithelium.

The exumbrellar epithelium of the hydromedusa, Euphysa japonica, is composed of a single layer of broad (70 micrometers), thin (1--2 micrometers) cells which are joined by gap junctions and simple appositions. Although the epithelium lacks nerves, it is excitable; electrically stimulating the epithelium initiates a propagated action potential. The average resting potential of the epithelial cells is -46 mV. The action potential, recorded with an intracellular electrode, is an all-or-nothing, positive, overshooting spike. The epithelial cells are electrically coupled. The passive electrical properties of the epithelium were determined from the decrement in membrane hyperpolarization with distance from an intracellular, positive current source. The two-dimensional space constant of the epithelium is 1.3 mm, the internal longitudinal resistivity of the cytoplasm and intercellular junctions is 196 omega cm, and the resistivity of both apical and basal cell membranes is greater than 23 k omega cm2. Although the membrane resistivity is high, the transverse resistivity of the epithelium is quite low (7.5 omega cm2), indicating that the epithelium is leaky with a large, transverse, paracellular shunt.

Action Potentials↗
PubMed1987

The role of changes in cell shape and contacts in the regulation of cytoskeleton expression during differentiation.

As a model for investigating gene regulation in relation to cell and tissue morphogenesis, we studied the expression of the adherens junction proteins, vinculin, alpha-actinin and actin, and that of desmosomal junctions containing the desmoplakin-cytokeratin complex, in response to changes in cell contacts and configuration. In monolayer or suspension cultures of kidney epithelial cells we found high levels of synthesis of cytokeratin and desmoplakin where extensive cell-cell contacts were established. In contrast, cells in sparse monolayers had high levels of the vimentin-type intermediate filaments, but very low levels of cytokeratins and desmoplakin I. Whereas in kidney epithelial cells all cytokeratins were coordinately regulated in response to changes in culture conditions, in mammary epithelial cells a new 45 X 10(3) Mr cytokeratin was induced in dense monolayer and suspension cultures. By treating cells with TPA, intercellular junctions were rapidly disrupted and expression of cytokeratin and desmoplakin was dramatically reduced; however, vimentin expression was not affected. In mammary epithelial cells only synthesis of the 45 X 10(3) Mr cytokeratin was reduced in TPA-treated cells. Thus the synthesis of the cytokeratin-desmoplakin complex was coordinately regulated in response to changes in cell-cell contact and cell shape in a way that is compatible with the organization of these cells in vivo. The relationship between the organization and expression of adherens junction proteins and their role in the acquisition of the differentiated phenotype was studied in fibroblasts and in differentiating ovarian granulosa cells. The synthesis of vinculin in cultured fibroblasts increased dramatically when the cell culture density was high, concomitant with the establishment of extensive cell-substratum and cell-cell contacts of the adherens type. When fibroblasts were plated on substrata of varying adhesiveness, to modulate cell shape from a flat and well-spread to a poorly adherent spherical shape, there was a relationship between vinculin organization and expression: vinculin synthesis decreased dramatically in round cells. The differentiation of freshly isolated ovarian granulosa cells (as measured by production of high levels of progesterone) in response to gonadotropic hormones was followed by dramatic changes in cell shape and organization and expression of adherens junction proteins. Cell shape changed from a flat fibroblastic type to a spherical one, with a reduction in vinculin-containing plaques and the disappearance of actin-containing stress fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Characterization of gap junctional communication-deficient mutants of a rat liver epithelial cell line.

To understand the mechanism(s) regulating gap junctional communication, we isolated gap junctional intercellular communication-deficient (GJIC-) mutant clones of a rat liver epithelial cell line, WB F-344, which is hypoxanthine-guanine phosphoribosyl transferase deficient (HGPRT-). The cells were exposed to a mutagenesis regimen and cocultured with the wild type HGPRT+ cells. Four GJIC- and one positive clones were characterized in the present study. Northern analysis of RNA isolated from both mutant and parental cells showed a single RNA species of about 3.0 kb which hybridized to connexin43 (Cx43) cDNA. Western blot analysis confirmed the expression of this junctional protein in all these clones. However, in the GJIC- clones the slowest migrating band corresponding to a hyperphosphorylated form, P2, of Cx43 protein (approximately 46 kDa) was absent suggesting that loss of this phosphorylated form of Cx43 may be involved in the failure of the mutants to establish cell-cell communication. Immunofluorescence analysis of the mutants did not reveal any differences in the distribution and localization of Cx43 between GJIC+ and GJIC- clones suggesting that the loss of phosphorylation did not affect the membrane association of this protein. Taken together, these data suggest that one mechanism for the loss of communication in these GJIC- mutants may be the consequence of a change in the intrinsic phosphorylation state of Cx43 protein.

Animals↗
PubMed2006

Choroid plexus papilloma in a Scottish highland cow.

Choroid plexus papilloma, a rare tumour in bovids, arising from the roof of the third ventricle was diagnosed at necropsy in a 4-year-old Scottish Highland cow. The animal presented with a 2-month history of progressive ataxia, ventromedial strabismus, and hyperaesthesia. Neoplastic epithelial cells were positive immunohistochemically for pancytokeratin and S-100, and negative for GFAP. Ultrastructurally, epithelial cells were characterized by intercellular junctions, zonulae adherens, and zonulae occludens. Neither cilia nor basal bodies were identified. The gross, microscopical, immunohistochemical and ultrastructural findings were consistent with those of a choroid plexus papilloma.

Animals↗
PubMed1975

Electron microscopic study on permeability of coronary artery wall of normotensive rabbits using horseradish peroxidase as a tracer.

In order to study the pathways in the coronary artery vessel wall horseradish peroxidase (HRP) was used as a tracer. HRP was injected directly in the left ventricle of rabbit hearts. 30 seconds after intracardial injection of HRP in normotensive rabbits, peroxidase reaction products could be located in random areas of the coronary arterial system and could then be observed in all three layers of the coronary walls at these locations. HRP reaches the subendothelial space of the coronary arteries along two pathways: through intercellular junctions and by plasmalemmal vesicles. Through the fenestations of the internal elastic lamina HRP reaches the media and can be detected between the media smooth muscle cells. The influence of the methodology on the results is discussed.

Animals↗
PubMed1996

Regulation of thrombin-mediated endothelial cell contraction and permeability.

A variety of physical forces exist in a dynamic equilibrium in the vascular endothelium (EC) monolayer and serve to maintain EC responsiveness while preserving the integrity of the EC monolayer and barrier properties. Thrombin has potent effects on EC permeabilities disrupting the equilibrium between tethering forces (cadherins, focal adhesion plaque) and forces that increase centripetal tension primarily via myosin light chain (MLC) phosphorylation. Like other EC effects, thrombin-induced MLC kinase (MLCK) activation is dependent upon receptor proteolysis, Ca2+ mobilization, and activation of protein kinase C (PKC). While EC gap formation is central to barrier dysfunction and dependent upon activation of MLCK, (which phosphorylates MLC) an obligatory event in smooth muscle cell contraction, little is known regarding the events that reverse inflammatory responses, halt the contractile response, and initiate relaxation. However, as these events likely include MLC dephosphorylation, further examination of the processes that regulate MLC protein phosphatase activity, focal intercellular junctions, and extracellular matrix adhesions is needed. These investigations should yield new information as to how receptor occupancy is transduced into specific cellular responses, such as increased permeability, which promotes pathological vascular processes such as tissue edema formation and organ dysfunction.

Animals↗
PubMedDate not supplied

Ultrastructural features of vocal fold nodules and polyps.

Earlier investigation did not show any clear distinctive pathological features that differentiate vocal polyps from vocal nodules. The light microscopic distinctive features between the two lesions point in the direction of a more pronounced epithelial reaction and a more fibrous stromal change in vocal nodules. In the present study, 19 vocal polyps and 11 vocal nodules were subjected to ultrastructural examination aiming at finding distinctive features that may be pathognomic for each of these clinically distinct entities. The study was carried out by EM Philips 400 T. The results demonstrate that nodules showed epithelial changes in the form of gaping of the intercellular junctions and absence of the basal lamina in parts. These changes are less apparent in polyps, whereas the stromal changes are more pronounced and varied. The significance of these results and their interpretation are discussed.

Adult↗
PubMed1983

The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart.

An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed. Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma. Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.

Animals↗