Sponge cell aggregation

Summary Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium.Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca²⁺; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of watercontaining channels by rearrangement in the secondary aggregates.On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, glucuronidase, ß-glucuronosyltransferase, ß- ß-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated ß-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin.The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.Abbreviations AF aggregation factor - CPP circular proteid particle - AR aggregation receptor - aAR anti-aggregation receptor - CMF calcium- and magnesium-free artificial sea water - ASW calcium- and magnesium-containing artificial sea water This paper is dedicated to PROF. DR. R. K. ZAHN on the occasion of his 60^th birthday.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

Purification and characterization of a species-specific aggregation factor in sponges

The events during re-aggregation of sponge cells, dissociated in Ca²⁺- and Mg²⁺-free artificial sea water, containing trypsin, can be subdivided into three phases. The first event is the formation of primary aggregates with a diameter of 82 μm. It is Ca²⁺- and Mg²⁺-dependent and insensitive towards trypsin and puromycin even at high concentrations. The formation of secondary aggregates with a diameter greater than 1 000 μm, is initiated by an aggregation factor. This factor can be separated from the formed elements with calcium- and magnesium-free artificial sea water. Its action is Ca²⁺- and Mg²⁺-dependent, temperature-independent and insensitive to puromycin. The last event is the reconstitution of functional aquiferous systems in the aggregates.The aggregation factor which was found to be species-specific could be purified 158-fold. Its functional group seems to be a protein probably with polar amino acids in critical positions. Secondary aggregates generated with the aggregation factor show high viability. Some evidence is presented that the aggregation factor may be an annular particle with a circular contour length of 3 500 Å with about 25 filaments attached to it.     

Various topochemical arrangements of sialic acids on human erythrocytes as detected by partition in aqueous two-polymer phase systems

Human and rabbit red blood cells were subjected to partition in an aqueous, buffered Ficoll-Dextran two-phase system. The effect of neuraminidase treatment on the cell partition behaviour was examined and compared with the amount of sialic acids released from the cell surface and with the change in the electrophoretic mobility of the cells. The data obtained in the study indicate that there are two main groups of sialic acids differing in their topochemical arrangement on the human erythrocyte surface, and their relative hydrophobicity was evaluated. The results obtained in the case of rabbit red cells seem to indicate that sialic acids present on the cell surface are not the only major ionogenic surface components as is the case for human red cells. The data obtained support the assumption that the membrane surface charge is the determinant of cell partition only as a factor affecting the relative hydrophobicity of the cell surface.     

Shape changes of the human red cell studied by aqueous two-phase partition

Aqueous two-phase systems have been used to study the human red cell during metabolically induced shape changes. When the discoid character of the cells was lost in favour of echinocytic forms, the partition increased both in charge-sensitive and in charge-insensitive two-phase systems. Reversal of the shape transformation by ATP repletion not only led to shape recovery but also restored the initial partition. Therefore it is apparent that red cells exhibit a shape-dependent partition behaviour. As the partition is dependent on surface properties (such as charge and hydrophobicity) of the partitioned material, the results show that the shape changes caused rearrangement of the membrane and thereby exposure of or greater accessibility of binding groups on the cell surface. The similar partition behaviour in the charge-sensitive and charge-insensitive phase systems show that the increased partition was caused mainly by increased hydrophobic interactions between the cells and the upper phase. The observed partition behaviour therefore suggests that the echinocytic cells acquire a higher affinity for the upper phase by repacking the lipid bilayer or at least the outer leaflet into a less efficient packed and thus more fluid structure.     

Internal reference standards in ionic regulation and the predictability of ionic concentrations in animals

• 1.1. The regulation of ions at similar concentrations in most individuals of a species suggests the existence of internal reference standards.
• 2.2. Few have been identified, but many probably relate to cell membrane properties, including potentials, surface charge densities and equilibrium constants of receptor molecules.
• 3.3. Solubility may sometimes determine the product [Ca²⁺][CO₃²⁻].
• 4.4. Reference standards must generally each relate to more than one ionic species.
• 5.5. For some concentrations, including osmolality, there may be no direct reference standard.

     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

The transport of potassium through lipid bilayer membranes by the neutral carriers valinomycin and monactin

Summary Stationary conductance experiments on neutral and negatively charged bilayer membranes in the presence of valinomycin or monactin agree with a recently proposed carrier transport model, which is common to both carrier types. This model assumes an interface reaction between a cation from the aqueous solution and a carrier molecule from the membrane phase to establish charge transport across the interface. The transport across the membrane interior is described by some kind of Eyring model. The discussion of the current-voltage characteristic, the dependence of membrane conductance on the carrier and K⁺ concentrations, and the comparison with appropriate experiments allow correlation of the different rate constants of the transport model. The results show that the rate constants partly depend on the surface charge of the membranes. This dependency can be described by introducing the Gouy-Chapman theory for charged surfaces into the transport model.It was found that the carrier molecules could be added either to the aqueous phase or to the membrane-forming solution. The quantitative treatment of this phenomenon gives an evaluation of the partition coefficient of the carrier molecules between the membrane bulk phase and water.     

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors ^1,2 and providing growth promoting adhesion molecules ³ and extracellular matrix molecules ⁴. In addition they myelinate the demyelinated axons at the site of injury ⁵.However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes ^6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules ^8-13. In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer ^14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) ^16,17, FGF/Heparin ¹⁸, Eph/Ephrins¹⁹.This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.Download video file.^{(64M, mov)}     

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.     

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.Mast cells play pivotal roles in inflammation and immediate-type allergic reactions by secreting biologically active substances including histamine, eicosanoids, proteolytic enzymes, cytokines, and chemokines. The antigen-induced aggregation of the high affinity IgE receptor (FcϵRI)² expressed on the cell surface triggers the degranulation of mast cells. FcϵRI has a tetrameric structure comprised of an IgE binding α-chain, a β-chain, and a disulfide-linked γ-chain dimer (1). The aggregation of FcϵRI by means of multivalent antigen-IgE complexes activates cytosolic Src protein-tyrosine kinases, such as Fyn and Lyn, which then regulate the activation of mast cells (2). Fyn kinase plays a key role in mast cell degranulation and in cytokine production by regulating Gab2 and phosphatidylinositol 3-kinase (3). Phosphorylated Lyn activates immunoreceptor tyrosine-based activation motifs of the β- and γ-chains, and the phosphorylated immunoreceptor tyrosine-based activation motifs of the γ-chain phosphorylate Syk kinase. Thereafter, a number of other signaling and adaptor molecules, such as phospholipase Cγ and protein kinase C (PKC), are phosphorylated (4). Phospholipase Cγ catalyzes the generation both of inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate is an inducer of intracellular Ca²⁺ mobilization, which is critical for degranulation, and diacylglycerol is an activator of PKC (5). Activated PKC is translocated from the cytosol to the plasma membrane fraction. PKC regulates many functions of mast cells, including leukotriene generation, cytokine synthesis, and degranulation (6, 7).Many studies have provided evidence that lipid rafts are involved in the activation of intracellular signaling molecules mediated by FcϵRI, the T cell receptor, the B cell receptor, and other cell surface receptors (8, 9). Lipid rafts are originally defined as microdomains in terms of their resistance to solubilization by non-ionic detergents such as Triton X-100, and are enriched in sphingolipids and cholesterol (10). Because numerous cell surface receptors and palmitoyl-anchored signaling molecules, including Src family tyrosine kinases, are associated with lipid rafts, it has been suggested that lipid rafts function as platforms regulating the induction of signaling pathways. Aggregated, but not non-aggregated, FcϵRIs are localized in lipid rafts fractionated by sucrose density gradient ultracentrifugation of detergent-treated cells (11, 12). The translocation of FcϵRI to lipid rafts is the key event that initiates the degranulation.Carotenoids are a class of widespread natural pigments that have multiple functions (13). Dietary carotenoids have been associated with a decreased risk for certain types of immune diseases, such as asthma and atopic dermatitis. Consumption of β-carotene suppresses the production of specific IgE and IgG₁ and decreases antigen-induced anaphylactic responses due to an improvement of the T_h1-T_h2 balance (14). Furthermore, β-carotene blocks nuclear translocation of the NF-κB p65 subunit, which correlates with the prevention of IκBα phosphorylation and degradation (15). It has been reported that fucoxanthin, a major carotenoid of edible brown algae, shows an anti-inflammatory effect on endotoxin-induced uveitis by decreasing the production of prostaglandin E₂ and tumor necrosis factor-α (16). Astaxanthin, found in the red pigment of crustacean shells and salmon, also has anti-inflammatory effects due to its suppression of NF-κB activation (17, 18). It has been assumed that these anti-inflammatory activities of carotenoids are due to their antioxidant activity. However, there is no information to date about the direct effect of carotenoids on the degranulation of mast cells.In the present study, we investigated the effects of carotenoids on antigen-induced degranulation of RBL-2H3 cells and mouse bone marrow-derived mast cells. In addition, to elucidate the mechanism of the modulation of degranulation by carotenoids, we focused on FcϵRI-mediated signaling in mast cells.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.     

Reaggregation of sponge cells: Specificity and recovery of adhesion

Summary The involvement of Concanavalin A (ConA), -methylmannose and glucuronic acid in the reaggregation of fresh water sponge cells has been tested. Glucuronic acid has been found to be the potent inhibitor and the indication is that this group is involved in the binding of aggregation factor to the cell surface. When cells were washed with EDTA and periodate to inactivate the aggregation factor, a lag phase of 5–6 h was required for the cells to recover the adhesive capacity. Puromycin does not inhibit this recovery process of EDTA washed cells. Con A, -methylmannose and glucuronic acid interfere with this recovery of adhesion and the cells remain nonaggregative, probably due to their interference with the insertion of aggregative molecules on the cell surface.