Non-adhesive Cells Research Articles

Type 1 fimbriae of Salmonella enterica serovar Typhimurium bind to enterocytes and contribute to colonization of swine in vivo.

Salmonella enterica serovar Typhimurium strain 798 is a clinical isolate from a pig and is known to be able to cause persistent, asymptomatic infections. This strain also is known to exist in two phenotypes (adhesive and nonadhesive to enterocytes) and can switch between the two phenotypes at a rate consistent with phase variation. Cells in the adhesive phenotype are more readily phagocytosed by leukocytes than nonadhesive cells. Once in a leukocyte, adhesive-phase cells survive while nonadhesive-phase cells die. In the present study, nonadhesive mutants were obtained with the transposon TnphoA. A nonadhesive mutant was selected for study and was shown by electron microscopy not to produce fimbriae. The gene encoding the adhesin was cloned and sequenced. Based on its sequence, the adhesin was shown to be FimA, the major subunit of type 1 fimbriae. The nonadhesive mutant was attenuated in its ability to colonize both mouse and pig intestines, but remained capable of systemic spread in mice. The nonadhesive mutant was phagocytosed to the same extent as parental cells in the adhesive phase and then survived intracellularly. These results demonstrated that type 1 fimbriae were important for attachment to enterocytes and promoted intestinal colonization. However, they were not important in promoting phagocytosis or intracellular survival.

Synthesis of functionally graded MgCO3 apatite accelerating osteoblast adhesion.

As a means of improving the biological properties of materials for use as bone substitutes, functionally graded carbonate apatite containing Mg, FGMgCO3Ap, was synthesized at 60 degrees C and pH 7.4 using a gradient magnesium supply system. X-ray diffraction analysis of FGMgCO3Ap showed a poorly crystallized apatitic pattern, similar to that of human bone. ESCA analysis clearly showed the negative gradient distribution in Mg1s intensity (atomic concentration) of magnesium from the crystal surface toward the inner core. When the FGMgCO3Ap crystals were mixed with collagen, the resulting FGMgCO3Ap-collagen composite, irradiated with UV light for 4 h, retained their features in the saline solution. After washing away the nonadhesive cells, a cell adhesion assay showed that the optical density of the FGMgCO3Ap-collagen composite was higher than that of the CO3Ap-collagen composite. SEM observation showed that the osteoblast-like cells adhered well to the surface of the FGMgCO3Ap-collagen composite. Staining with hematoxylin-eosin and alizarin red confirmed the existence of a great many more cells and a thicker extracellular matrix layer on the FGMgCO3Ap-collagen composite than on the CO3Ap-collagen composite. This result demonstrated the acceleration effect of magnesium ions on osteoblast adhesion on the FGMgCO3Ap-collagen composite.

A simple flow-through micro-chamber for handling fragile, small tissue explants and single non-adherent cells.

The flow-through chambers described are easily assembled in the laboratory with no need for sophisticated or expensive equipment. They allow for treating micro-explants or single non-adhesive cells with different solutes for defined times by easy and quick changes of the medium in the chambers. The specimen can be fixed, immunostained and observed microscopically within the chamber. During these procedures the cells are not exposed to undue mechanical stress and fragile intercellular structures are well preserved.

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro.

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

Tat-mediated delivery of antibodies into cultured cells.

Many questions concerning the function of the cytoskeleton can be addressed using functional antibodies either in in vitro assays or in whole cell systems. Ultimately, the function of a component has to be studied within intact cell systems. A problem, which one has to be overcome, is the delivery of the antibody into the cells. The delivery of antibodies can be a particular problem either, if large numbers of cells will be analyzed or if one wants to study adhesive as well as non-adhesive cells. Several approaches to introduce proteins and other components into cells have been published, such as electroporation (), scrape loading (), or delivery via liposomes (). Among these the most widespread are microinjection and electroporation. While microinjection is routinely used for adhesive cells, this method will be very time consuming when large numbers of cells have to be studied. Also, it is not feasible to employ this technique for small cells in suspension. Electroporation, on the other hand, has been employed successfully to introduce various components in to cells in suspension. Chang (,) has also developed an electroporation method for adhesive cells using an oscillating electric field; still a fare number of cells die during this procedure.

Attachment of the yeast Rhodosporidium toruloides is mediated by adhesives localized at sites of bud cell development.

The basidiomycetous yeast Rhodosporidium toruloides (anamorph, Rhodotorula glutinis) is a common phylloplane epiphyte with biocontrol potential. To understand how R. toruloides adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att-) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type R. toruloides was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att- mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of R. toruloides to both polystyrene and barley leaf surfaces.

Plasmatocyte spreading peptide does not induce Microplitis demolitor polydnavirus-infected plasmatocytes to spread on foreign surfaces.

Capsule formation by the moth Pseudopulsia includens requires that plasmatocytes change from being nonadhesive cells in circulation to strongly adhesive cells capable of attaching to the foreign target and one another. This change in adhesive state is induced by Plasmatocyte Spreading Peptide (PSP1); a 23 amino acid peptide isolated from P. includens plasma. Plasmatocytes from hosts parasitized by Microplitis demolitor remain in a nonadhesive state after infection by Microplitis demolitor polydnavirus (MdPDV). This alteration in plasmatocyte function prevents P. includens from encapsulating the developing parasitoid. In the current study, we examined whether MdPDV infection eliminates PSP1-responsive plasmatocytes from circulation or disrupts the ability of PSP1 to induce adhesion and spreading of plasmatocytes to foreign surfaces. In vivo experiments revealed that infection of P. includens by MdPDV induced an increase in the total number of hemocytes in circulation but reduced the proportion of hemocytes in circulation that were plasmatocytes. However, plasmatocytes normally capable of responding to PSP1 were not eliminated from circulation. Both in vivo and in vitro experiments indicated that plasmatocytes inoculated with MdPDV lost the capacity to respond to PSP1 4-6 h post-infection. Infection of P. includens with MdPDV reduced expression levels of prepro-PSP1 mRNA in hemocytes but did not appear to alter expression levels in fat body.

Phase variable switching of in vivo and environmental phenotypes of Salmonella typhimurium.

Previously it was shown that S. typhimurium strain 798, which is known to cause persistent asymptomatic infections in pigs, exists in two phenotypes. One phenotype, which is called adhesive, was shown to produce pili, is adhesive to porcine enterocytes, is readily phagocytized, and then survives intracellularly in phagocytes. The other phenotype, termed non-adhesive, does not produce pili, does not attach to enterocytes, is phagocytized less efficiently, and does not survive within the phagocyte. Cells in each phenotype can freely switch to the other phenotype at a fairly high frequency and thus the shift between each phenotype is phase variation. Further analysis of these phenotypes identified 4 additional characteristics that were co-regulated by phase variation. The first is the enterocyte-specific adhesin, which was shown to be type 1 fimbriae. Mutations in fimA, the major pilin molecule, led to a decreased ability to colonize the gut of pigs and mice. The second characteristic is O-antigen production. Adhesive cells produce a long O-antigen (up to 18 subunits) while non-adhesive cells do not (only 1-2 subunits). The long O-antigen produced by the adhesive cells leads to resistance to serum and appears to be the result of phase variable expression of rfaL. A third locus, ebu, has been identified based on differential color production of colonies growing on Evans blue-Uranine plates. The relationship of this trait to in vivo survival or virulence is not known but ebu is genetically related to a family of transcriptional activators. The fourth locus, prv is located on the virulence plasmid and a mutation in prv results in delayed time to death in mice. It is hypothesized that the adhesive phenotype is the in vivo, virulent form, while the non-adhesive phenotype is the environmental, avirulent form. By modulating the fraction of cells in each phase, persistent asymptomatic infections can be promoted.

Protein zero, a myelin IgCAM, induces physiologically operative tight junctions in nonadhesive carcinoma cells

Protein zero, a myelin IgCAM, induces physiologically operative tight junctions in nonadhesive carcinoma cells

Protein zero, a myelin IgCAM, induces physiologically operative tight junctions in nonadhesive carcinoma cells.

In the peripheral nervous system, protein zero (P0), a homophilic immunoglubulin cell adhesion molecule, mediates adhesion of Schwann cell membranes as they enwrap axons and generate compact myelin. Although P0 is naturally only expressed in peripheral myelin, it can behave as a vigorous adhesion molecule in a variety of cell types (Filbin et al. [1990] Nature 344:871-872; Schneider-Schaulies et al. [1990] J Neurosci Res 27:286-297; Doyle et al. [1995] J Cell Biol 131:465-482) and can thus be characterized as an obligatory adhesion molecule. Previously, we showed that when HeLa, a cervical carcinoma cell line devoid of epithelial junctions, is forced to express P0, strong cell-cell adhesion is induced, proteins associated with junctional elements are upregulated, and ultrastructurally tight junctions, adherens junctions, and desmosomes become apparent (Doyle et al., 1995). In this report, we assessed whether the tight junctions were physiologically operative in P0 HeLa expressors. Consistent with the presence of operative tight junctions, we found that P0 expressors in monolayers maintained endogenous proteins in their apical and basolateral plasma membrane subdomains. Furthermore, these cells generated a higher transepithelial resistance than did control HeLa cells, which is indicative of the formation of an effective intercellular permeability barrier.

Regulation of endothelial cell and platelet receptor-ligand binding by the 12- and 15-lipoxygenase monohydroxides, 12-, 15-HETE and 13-HODE

In previous studies, we reported that vascular wall cells such as endothelial cells metabolize linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) via the 15-lipoxygenase pathway. Endothelial cell 13-HODE levels vary inversely with endothelial cell reactivity to platelets, which, in turn, varies directly with the expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells. We and others have also found that tumour cell adhesivity is dependent, in part, upon the relative amounts of intracellular 13-HODE and the arachidonic acid monohydroxide(s), 12- and/or 15-hydroxyeicosatetraenoic acids (12-, 15-HETE). In addition, we and others have found that platelet adhesivity is dependent upon the intraplatelet level of its major lipoxygenase metabolite, 12-HETE. Finally, we have demonstrated that 13-HODE and VnR co-localize in nonadhesive endothelial cells but dissociate following endothelial cell injury, at which time, the VnR relocates on the endothelial cell apical surface. These data suggest to us that lipoxygenase-derived monohydroxides regulate the ability of various receptors to recognize their specific ligands. The latter data also suggest that these monohydroxides act directly by a physiochemical mechanism. The present study supports this possibility. Thus, we demonstrate that 13-HODE downregulates VnR binding with vitronectin (Vn) > fibronectin (Fn) > fibrinogen (Fgn), whereas 12- and 15-HETE upregulate specific VnR/ligand binding, using purified VnR/liposomes and purified ligands in an adhesion assay; and that 12- and 15-HETE upregulate GPIIb/IIIa:liposome binding of Fgn > Fn > Vn. We conclude that cell-specific monohydroxides influence cell-specific receptor-ligand binding directly through a physiochemical mechanism.

Altered Cell Adhesion Activity by Pervanadate Due to the Dissociation of α-Catenin from the E-Cadherin·Catenin Complex

Leukemia cells (K562) that grow as non-adhesive single cells and have no endogenous cadherin were transfected with an E-cadherin expression vector, and cell clones stably expressing E-cadherin on their surface were established. The expression of E-cadherin induced the up-regulation of catenins, and E-cadherin became associated with catenins. The transfected cells grew as floating aggregates. Cell aggregation was Ca2+-dependent and was inhibited by E-cadherin antibodies. The aggregates dissociated into single cells on the addition of pervanadate. Pervanadate caused a dramatic augmentation of the phosphorylation of E-cadherin, beta-catenin, and gamma-catenin (plakoglobin), but alpha-catenin was not detectably phosphorylated. After pervanadate treatment, beta-catenin and gamma-catenin migrated more slowly on gel electrophoresis, suggesting changes in their conformations due to eventual changes in their phosphorylation levels. In the treated cells, a significant amount of alpha-catenin was dissociated from the E-cadherin.catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with alpha-catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of alpha-catenin from the complex underlies the observed E-cadherin dysfunction.

Cytotoxic activity in culture filtrates from the entomopathogenic fungus Beauveria sulfurescens

The hyphomycete fungus Beauveria sulfurescens secretes into the culture medium several components toxic for Galleria mellonella larvae. Concentrated and dialyzed (cutoff 12,000) B. sulfurescens culture filtrate also contains a highmolecular-weight compound cytotoxic for the Mamestra brassicae cell line. A fractionation in two chromatographic steps separated a cytotoxic fraction (IC50= 1.15 ± 0.05 μg/ ml), which was poorly toxic to larvae (LC50 = 150 ± 10 μg/ ml) and an insecticidal fraction (LC50 = 2.1 ± 0.1 μg/ml) devoid of detectable cytotoxicity. The two kinds of activity, i.e., cytotoxicity in vitroand toxicity in vivo are thus independent. The cytotoxic activity, initially detected on a M. brassicae cell line, also affected two other insect cell lines (G. mellonella and Spodoptera frugiperda). The cytotoxic fraction provoked not only the inhibition of cell proliferation, but also cell death. Adhesive mammalian cell lines were not sensitive. Nonadhesive mammalian cell lines of murine myeloma and hybridomas were poorly sensitive.

The Drosophila membrane receptor Toll can function to promote cellular adhesion.

The product of the Toll gene is a membrane protein required for the formation of dorso-ventral polarity during early embryogenesis in Drosophila melanogaster. It acts together with the other dorsal group gene products to specify a nuclear gradient of dorsal morphogen in the syncytial blastoderm stage embryo. Here we report the presence in Toll protein of additional sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). We propose that these sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. In addition, we show that expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules. Taken together these findings suggest that the biochemical function of Toll protein is more closely analogous to that of Gp1b than previously thought.

Changes in DNA and surface properties of peripheral blood leukocytes in monkeys after whole-body ?-irradiation

Changes of Macaca nemestrina and Rhesus blood DNA have been studied up to 5 days after the total uniform gamma-irradiation in doses 6.2 and 6.5 Gy. The content of nucleotide ATrich DNA has been evaluated in the fractions of leucocytes with the various surface adherence properties. The dynamics of the content nucleotide AT-DNA and blood leucocytes were similar at the both monkey species. The evaluation of structural state DNA in the blood nucleotide with the fluorescent dyes (ethidium bromide and 4; 6-diamidine-2-phenylindole) demonstrated that the important changes in the polynucleotide structure occurred from 6 to 24 h after irradiation and maintained up to 5 days. Adhesive capacity changes were reversible but they preceded the DNA structural changes. At 24 h postirradiation non-adhesive cells with relative higher AT-DNA content were found.

Developmentally regulated cell-cell adhesion in Dictyostelium purpureum is mediated by a glycoprotein synthesized in nonadhesive cells

Upon starvation the cellular slime mold, Dictyostelium purpureum, develops a form of cell-cell adhesion aiding in the formation of large multicellular aggregates, which are capable of further differentiation. The molecule that mediates this adhesion is a glycoprotein of M r approximately 40,000. The protein shares a common carbohydrate epitope with another well-characterized cell adhesion molecule from Dictyostelium discoideum, contact sites A, but the polypeptides to which it is attached differ for each species. Although mediating a developmental form of adhesiveness, the protein is synthesized in vegetative cells at a time when they do not adhere. Most of the vegetative protein is associated with cell membranes and appears to be on the surface of these cells. The protein is compared to other cell adhesion molecules from other species of cellular slime molds, and possible explanations for its inability to function in vegetative cells are discussed.

Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains

A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones of substrate adherent mammalian cells at a rate of one clone/10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems.

Enzymic dissection of embryonic cell adhesive mechanisms. III. Immunological identification of a component of the calcium-dependent adhesive system of embryonic chick neural retina cells.

Immunological probes and cell-surface biochemical analysis are combined to identify a cell-surface glycoprotein, which appears to be involved in the formation of calcium-dependent adhesions among embryonic chick neural retina cells. Comparison of radioiodinated cells shows that adhesive-competent cells have at their surface two glycoproteins not present on non-adhesive cells. These two components (with Mr 130 x 10(3), pI 4.8 and Mr 70 x 10(3), pI 4.8) are immunoprecipitable with adhesion-blocking antisera. A fraction derived from retina-conditioned medium, containing one major immunoprecipitable component, neutralizes the inhibitory activity of aggregation-inhibiting Fab' fragments and competes in immunoprecipitation for the two glycoprotein components. The evidence suggests that one or both of these components is involved in the formation of calcium-dependent adhesions among embryonic chick neural retina cells.

Isolation of two types of platelets by collagen-sepharose conjugate and elucidation of their roles in adhesion and aggregation

Approximately 50% of washed platelets from bovine blood adhered to collagen-Sepharose conjugate in the presence of Ca 2+ and did not adhere to bovine serum albumin and ovalbumin. The extent of adhesion was increased neither by decreasing the number of platelets applied, nor by increasing the column volume. We have thus shown that platelets in the normal blood may be subdivided into two groups depending on their adhesiveness to collagen-Sepharose, adhesive and non-adhesive cells. The adhesive cells did not seem to aggregate themselves, while the non-adhesive cells aggregated quite normally. Furthermore, we were able to reveal that adhesive cells could aggregate only with non-adhesive cells. We propose that these two types of platelets played their respective roles sequentially to ensure the successful formation of thrombus in vivo .

Human lymphoblastoid cell variants defective in cell-cell adhesion.

Adhesion mutants were selected from a human lymphoblastoid cell line. Initially, cells were selected on the basis of survival in serum-free medium. Subclones that grow as single cells rather than macroscopic aggregates were selected from the serum-independent variant. The defect in cell-cell adhesion is stable over many generations and is not corrected by growth in serum or the presence of serum in the culture medium. Analysis of mixed cultures composed of adhesive cells and nonadhesive cells indicates that the two cell types do not interact to form mixed aggregations. Furthermore, those results suggest that the adhesion-deficient phenotype does not result from the production of a transferable inhibitor. In a previous study [Whipple, A.P., Dalvin, M. & Millis, A.J.T. (1978) Exp. Cell Res. 116, 457-461], we found that the growth rate in serum-containing medium is identical for the two classes of cells. This suggests that cell-cell adhesion is not a critical factor in the growth of these cells.