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R A Badley

Publications and source records attributed to R A Badley.

At least 37 records · Page 2Linked to original sources
PubMed1983

Histopathological diagnosis of hydatidiform mole.

Two microscopical methods are described which are useful for the identification of hydatidiform molar trophoblast. The first is an application of Nomarski's differential interference contrast microscopy which allows whole-mounted villi to be studied by optical sectioning. The second is an application of indirect immunofluorescence microscopy using a specific antiserum directed against the endothelial cell structural protein-known as vimentin.

Female↗
PubMed1983

Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts.

The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin are distributed generally with some reticular patterning in the early motile cells which lack the focal specializations, but as focal contacts and adhesions form these proteins become progressively recruited into the associated microfilament bundles. Only then do we see the marked depletion that has been reported earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage and bracing of cells to the substratum when they later become stationary.

Actinin↗
PubMed1983

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos.

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.

Animals↗
PubMed1983

Characterization of a detergent-resistant surface lamina in cultured human fibroblasts.

Treatment of cultured human fibroblasts with 0.5% Triton X-100 produces substratum-anchored cytoskeletal preparations consisting of cytoplasmic filaments, nucleus and a plasma membrane-derived surface lamina. The lamina was visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin (WGA) as a lace-like structure, extending throughout the cell domain. It displayed a different organization at the ventral and dorsal surfaces of the cell, partially coaligning with bundles of actin and myosin filaments at the dorsal cell surface. At the ventral surface vinculin patches appeared to be included in the surface lamina. Polyacrylamide gel electrophoresis, combined with lectin reactivity studies and lectin affinity chromatography, revealed a 140 kD sialoglycoprotein as the major glycoprotein component of the surface lamina.

Cell Membrane↗
PubMed1983

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation.

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.

Actins↗
PubMed1983

Microfilaments and intermediate filaments in epithelial cells transformed by murine sarcoma or leukemia viruses.

The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene.

Actin Cytoskeleton↗
PubMed1983

Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins.

The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact, characteristically in the form of short radial stitches, was examined in more detail using immunoelectron microscopy with colloidal gold as marker. This showed the close proximity of fibronectin to the cell membrane, with the ventral surface and fine cellular processes showing the heaviest labelling, and also revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was distributed as a circumferential bundle of filaments, with finer stands running radially to the edge. The latter were reminiscent of the radial fibronectin stitches and a spatial correspondence between fibronectin and actin was confirmed by double-label immunofluorescence which revealed many instances of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved in keratinocyte adhesion and is related to the internal organisation of these cells.

Actins↗
PubMed1982

Expression of intermediate filaments in soft-tissue sarcomas.

Monospecific antibodies and indirect immunofluorescence microscopy were used to investigate the presence of cytoskeletal intermediate filaments of the keratin, vimentin and desmin types in 43 soft-tissue sarcomas. The results showed that vimentin was present in the neoplastic cells of all types of soft-tissue sarcomas studied, whereas keratin was absent, the only exception being the epithelial-like cells in biphasic synovial sarcoma. The presence of desmin was confined to leiomyosarcomas and rhabdomyosarcomas, which usually showed desminpositivity. In addition, malignant fibrous histiocytomas occasionally displayed some desmin-positive cells. Thus, determination of intermediate filaments is of aid in the histogenetical diagnosis of tumors, because keratin-positivity largely excludes the possibility of a sarcoma, and desmin-positivity aids in recognizing leiomyosarcomas and rhabdomyosarcomas.

Desmin↗
PubMed1982

Organization of actin filaments in early chick embryo ectoderm: an ultrastructural and immunocytochemical study.

By combining transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunocytochemistry at both light and TEM levels the organization of actin in early chick ectoderm has been demonstrated. Every cell is encircled by a ring of actin filaments which are associated with apically situated intercellular junctions. In this way the actin ring of each individual cell is connected to that of surrounding cells and a continuous network is formed. As clearly shown by immunofluorescence and SEM studies on intact and Triton X-100 treated whole-mount preparations this network extends throughout the entire layer, interrupted only at the blastoderm margins and the primitive streak. A more diffuse arrangement was sometimes seen at cell bases. It is suggested that the network extending throughout the ectoderm gives it stability and cohesion and is important in gross morphogenetic movements involving the entire layer. The less-organized arrangement at cell bases is associated more with movements of individual cells.

Actins↗
PubMed1982

Alveolar rhabdomyosarcoma. Demonstration of the muscle type of intermediate filament protein, desmin, as a diagnostic aid.

Three cases of soft-tissue sarcomas with the characteristic histologic features of alveolar rhabdomyosarcoma, but lacking cytoplasmic cross-striations, were studied ultrastructurally and immunohistochemically to confirm the diagnosis and evaluate the histogenesis. The results showed that it was not possible to judge the skeletal muscle derivation of the cells at the ultrastructural level. However, immunohistochemically, the results of every case were positive for desmin-the muscle type of the intermediate filament protein. The results suggest that demonstration of desmin may be a helpful adjunct tool in the diagnosis of poorly differentiated alveolar rhabdomyosarcomas.

Adolescent↗
PubMed1982

Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent macrophages.

Indirect immunofluorescence microscopy was used to study the reorganization of contractile and cytoskeletal elements of human monocytes during their in vitro transition into macrophage-like cells. In freshly isolated monocytes, actin, myosin, alpha-actinin, and vinculin all had a diffuse distribution, whereas microtubules and vimentin-type intermediate filaments showed a distinct fibrillar organization. In culture, the cells differentiated within 1 week to two distinct cell types: bipolar fibroblastoid cells and flattened epithelioid cells. A conspicuous redistribution of actin, alpha-actinin, and vinculin from a diffuse cytoplasmic location into distinct punctate foci at the substratum-facing side of the cells was seen already at the early attachment phase of the spreading monocytes. In spread cells the punctate foci occupied the whole ventral surface of the cells, and in flattened, fully spread cells these proteins formed distinct punctate plaques at the under surface of the cells excluding however, the ruffle edge-like membrane regions. At the plaques, a close co-distribution of actin and vinculin was seen in double indirect immunofluorescence microscopy. Myosin showed a distinctly different reorganization during the transition process changing from a diffuse cytoplasmic location to a striated, surface-associated distribution. Interference reflection microscopy revealed large areas of close adhesion but no focal adhesion sites in spreading monocytes. Cytochalasin B treatment lead to a rapid distortion of the actin organization and resulted in clump-like cytoplasmic aggregates and nuclear paracrystals of actin concomitantly with the rounding up of the cells. Vimentin filaments were seen as a perinuclear aggregate until the acquisition of the fully spread morphology. At this stage both vimentin filaments and microtubules displayed a fibrillar appearance throughout the cytoplasmic domain. Treatment of the spread monocytes with antimitotic drugs caused a retraction of the cell from beyond the attachment plaques but had no effect on the punctate actin organization. The results show that in the monocyte-macrophage transition there is an extensive cytoskeletal reorganization that can be correlated with the different phases of the cell-to-substratum attachment process.

Actinin↗
PubMed1982

Rhabdomyoma: ultrastructural features and distribution of desmin, muscle type of intermediate filament protein.

A case of adult type rhabdomyoma in the neck region of a 77-year-old man is presented. In electron microscopy, some tumour cells showed irregularly orientated sarcomere formation with distinct Z-bands. All cells contained rod-like inclusions with attached thin cytoplasmic filaments. When stained with antibodies against desmin many tumour cells showed a diffuse peroxidase reaction, but the tumour also contained cells with cross striations. After phosphotungstic acid hematoxylin staining, dense cytoplasmic inclusions were seen; these structures showed strong affinity to antibodies against desmin. Our results apparently confirm the suggestion that the rod-like inclusions in rhabdomyoma cells are modified Z-bands. The desmin staining patterns in the present tumour may represent different stages in disorderly proliferation and degeneration of rhabdomyoma cells.

Aged↗
PubMed1981

Morphogenesis of human placental chorionic villi: cytoskeletal, syncytioskeletal and extracellular matrix proteins.

Immunocytochemical and ultrastructural methods were used to investigate the distribution of a family of structural proteins in the human placenta near term. These reveal the distribution of cytoskeletal and 'syncytioskeletal' components that may account for some of the more obvious micromorphological features of placental structure. In the syncytiotrophoblast a potentially supporting structure 'the syncytioskeletal layer' is described. It is an apparently continuous and complex polymeric network covering the villous tree, a surface of the order of 10 m2 in area in the full term placenta (Aherne & Dunnill 1966). It is suggested that this layer plays a part in morphogenesis of the villous tree.

Chorionic Villi↗
PubMed1981

Propidium iodide as a nuclear marker in immunofluorescence. I. Use with tissue and cytoskeleton studies.

Some examples are given of immunofluorescence with tissue sections and microtubular cytoskeletons of cultured cells where the fluorescent dye propidium iodide (PI) has been used as marker of nuclei. The emission wave length of IP is longer than that of fluorescein, making it possible to use several different and commonly available filter combinations. The use of nuclei as positional indicators is often a more suitable method than phase microscopy combined with immunofluorescence because of low background illumination against which morphology is viewed, circumventing the need for often expensive phase optics.

Antibodies↗
PubMed1981

Cooperativity of concanavalin A patching and its influence on cytoskeleton changes in fibroblast rounding and detachment.

We have studied the extent of concanavalin A (Con A) patching on spread fibroblasts as a function of increasing concentration of added Con A, and also the influence of this patching on subsequent cytoskeleton changes during exposure to trypsin and EGTA. Living cells do not patch at low concentrations of Con A (around 1 microgram ml-1) but progressively do so at higher levels (2-10 microgram ml-1), in very close parallel with a cooperative phase of binding characterized earlier. The proportion of visibly patched cells is at a maximum when this cooperative phase is complete and the correlation remains when cells are treated with azide of cytochalasin B to shift the concentration range over which both phenomena occur. All this evidence points to a common origin for cooperative binding and visible patching which is sensitive to the state of cellular actomyosin, and involves the mediation of actin-associated structures. As a consequence of Con A patching, cell attachment is preserved against the action of EGTA. On patched cells, this agent causes minor if any changes in cell shape and in the distributions of microtubules and 10-nm filaments, but actin stress fibres are dispersed. However, conditions appear to be retained for reassembly of the stress fibres because these can be caused to reappear within minutes after removal of EGTA when glucose, or Ca2+ or Mn2+ in the absence of glucose, is supplied in the medium. Trypsin caused only partial destruction of stress fibres in Con A-treated cells and did not promote detachment or dispersal of microtubules or 10-nm filament systems.

Animals↗