Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags

Reactive phosphorylcholine polymers, which can recognize biosynthetic cell-surface tags, were synthesized to control cell attachment. Human promyelocytic leukemia cells (HL-60) with unnatural carbohydrates as cell-surface tags were harvested by treatment with N-levulinoylmannosamine (ManLev). The attachment of ManLev-treated HL-60 cells to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with hydrazide groups was studied. HL-60 cells, which are nonadhesive, did not attach to any polymer surface without ManLev treatment. In contrast, ManLev-treated HL-60 cells attached to a poly[MPC-co-n-butyl methacrylate (BMA)-co-methacryloyl hydrazide (MH)] (PMBH) surface following 15 min of incubation. The cells that attached to the PMBH surface retained their native morphology and viability for 24 h of incubation. On the other hand, approximately half of the HL-60 cells that attached to the poly(BMA-co-MH) (PBH) surface died. These results suggest that MH units in the polymer act as anchors for cell attachment and MPC units help to preserve cell viability on a polymer surface. The coculture of ManLev-treated HL-60 and fluorescence-stained human uterine cervical cancer cells (HeLa) was carried out on polymer surfaces. ManLev-treated HL-60 cells specifically attached to the PMBH surface. In contrast, both HL-60 and HeLa cells were observed on the PBH surface. The control of cellular interactions with synthetic polymers may be useful for the future development of cell-integrated biosensors and biomedical devices.     

Evidence for covalent attachment of diphytanylglyceryl phosphate to the cell-surface glycoprotein of Halobacterium halobium.

In a previous study, we demonstrated the occurrence of novel proteins modified with a diphytanylglyceryl group in thioether linkage in Halobacterium halobium (Sagami, H., Kikuchi, A., and Ogura, K. (1995) J. Biol. Chem. 270, 14851-14854). In this study, we further investigated protein isoprenoid modification in this halobacterium using several radioactive tracers such as [3H]geranylgeranyl diphosphate. One of the radioactive bands observed on SDS-polyacrylamide gel electrophoresis corresponded to a periodic acid-Schiff stain-positive protein (200 kDa). Radioactive and periodic acid-Schiff stain-positive peptides (28 kDa) were obtained by trypsin digestion of the labeled proteins. The radioactive materials released by acid treatment of the peptides showed a similar mobility to dolichyl (C55) phosphate on a normal-phase thin-layer plate. However, radioactive hydrolysates obtained by acid phosphatase treatment co-migrated not with dolichol (C55-65), but with diphytanylglycerol on both reverse- and normal-phase thin-layer plates. The mass spectrum of the hydrolysate was also coincident with that of diphytanylglycerol. The partial amino acid sequences of the 28-kDa peptides were found in a fragment (amino acids 731-816) obtainable by trypsin cleavage of the known cell-surface glycoprotein of this halobacterium. These results indicate that the cell-surface glycoprotein (200 kDa) is modified with diphytanylglyceryl phosphate.     

Classification of stability behavior of bioreactors with wall attachment and substrate-inhibited kinetics

The biodegradation of pollutants in continuous operation when the microbial population exhibits wall attachment is studied. The proposed model for wall attachment assumes two morphological forms of the microbial cell connected by metamorphosis reactions with first order exchange kinetics. An analysis of the stability of the bioreactor, carried out using elementary principles of the singularity theory and continuation techniques, allows for classification in the multidimensional parameter space of the various stability behaviors exhibited by the reactor model, for both substrate-inhibited and Monod kinetics. The analysis also shows the enhanced stability behavior of the bioreactor due to wall attachment.     

The Biochemical characterization of syrian hamster cell-surface alloantigen

The first alloantiserum to be described in Syrian hamsters has been characterized for its ability to react with externally and internally radiolabeled antigens derived from normal hamster lymphoid cells. Utilizing conventional biochemical techniques, radioiodinated and³H-leucine labeled cellular extracts have been prepared, partially purified by lentil lectin affinity chromatography, and indirectly immunoprecipitated with experimental alloantisera. Analysis of the precipitated radiolabeled antigens by polyacrylamide gel electrophoresis in SDS has identified two prominent cell-surface proteins at 39,000 (p39) and 29,000 daltons (p29) on 2-mercaptoethanol reduced gels. Further analysis of the radiolabeled extract has demonstrated the existence of hamster cell-surface proteins at 43,000 and 12,000 daltons which are immunoprecipitated by a xenoantiserum directed against human ₂ microglobulin. Coelectrophoretic studies indicate the independent identity of these four species of hamster cellsurface proteins. These results suggest that between two hamster lines, derived from animals caught 40 years apart from different geographic locations in Syria, polymorphism of cell-surface antigens is restricted to p39 and p29 molecular species.     

一株高产细胞表面植酸酶酵母的实验研究

对一株高产细胞表面植酸酶酵母突变株WZ4菌的细胞植酸酶进行了研究。探讨了菌体生长与产酶的关系。结果表明:菌体在培养的前期植酸酶酶活很低,培养30h后酶活迅速增加,酶活在菌体生长的平衡期达到最大,该菌产植酸酶为非生长偶联型。在此基础了解WZ4菌细胞植酸酶的性质,实验表明:该酶的最适pH为5,最适温度为50℃,Km(以植酸钠为底物)为0666mmolL。通过磷对产酶影响因素的实验研究,初步得到WZ4菌产植酸酶受控于培养基中的磷浓度的结论,即最大产酶磷浓度为05mg100ml,当磷浓度大于10mg100ml时产酶被完全阻遏。     

The use of mathematical models for description of kinetics of attachment of human neutrophils to substrate in vitro

Kinetics of morphometrical parameters (perimeter, square, compactness) of human blood neutrophils attached to object glass is described by equations similar to the kinetical equations of irreversible reactions of the first reactions. The calculated values of morphometrical parameters of neutrophils in suspension and those attached to glass agree with the literature data. It has been found that compactness of neutrophils in the process of attachment is not changed. The time of completion of the process of attachment amounts approximately to 3 h.     

Microbial cell-surface display

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion. The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins. Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.     

The neurobiology of attachment

It is difficult to think of any behavioural process that is more intrinsically important to us than attachment. Feeding, sleeping and locomotion are all necessary for survival, but humans are, as Baruch Spinoza famously noted, "a social animal" and it is our social attachments that we live for. Over the past decade, studies in a range of vertebrates, including humans, have begun to address the neural basis of attachment at a molecular, cellular and systems level. This review describes some of the important insights from this work.     

The cell-surface distribution of alloantigens on the bovine erythrocyte

The topographical distribution of genetically defined alloantigens of the bovine erythrocyte was investigated by a serological absorption procedure known as the blocking test. Blocking was observed between the E′₁ and Y₂ antigens in theB system when these occurred in the same phenogroup. E′₁ was also blocked by G and Q′ in the same phenogroup and Y₂ was blocked by G. No blocking was observed between specificities residing either in different phenogroups or in different systems. We conclude that the several specificities in a givenB-system phenogroup are carried on a single molecule having multiple antigenic determinants, and that this arrangement is entirely consistent with the hypothesis that these factors are controlled by multiple alleles at a single locus.     

Spatial distribution of mammalian cells grown on macroporous microcarriers with improved attachment kinetics.

Vero and HepG2 cells were cultivated on macroporous gelatin microcarriers prepared by the calcium carbonate inclusion method. Cell attachment to these microcarriers was slow. For HepG2 cells the subsequent growth was poor. Modification of the microcarriers by incorporation of (diethylamino)ethyl-HCl improved HepG2 attachment and subsequent growth. Optical sectioning with confocal microscopy allowed visualization of the distribution of cells within microcarriers. In most microcarriers, cells were found to preferentially populate regions close to the external surface and some cavities in the interior. Despite the incomplete occupancy of the interior of the microcarriers, high cell concentrations were achieved.     

A stopped-flow fluorescence anisotropy method for measuring hormone binding and dissociation kinetics with cell-surface receptors in living cells

We have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1-15nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.     

Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fertilization studies in the hamster : The role of cell-surface carbohydrates

The nature of complementary binding sites on the surfaces of hamster gametes has been analysed using mono- and oligosaccharides, glycoproteins and glycosidases in an in vitro system. The binding of capacitated spermatozoa to the zona pellucida was inhibited by several mono- and oligosaccharides related to fucose, galactose, and acetylated amino sugars, but not by unrelated sugars. Several glycoproteins with prosthetic carbohydrate groups rich in or terminated by galactose or N-acetylglucosamine residues were also potent inhibitors of fertilization. Of all the glucoproteins tested, two plasma glycoproteins, α₁-acid glycoproteins (orosomucoid) and fetuin were most effective. In their native form they were non-inhibitory but their desialylated (galactoseterminated) forms completely prevented the sperm-zona binding. Agalacto-orosomucoid with N-acetylglucosamine terminals also inhibited fertilization. The treatment of capacitated spermatozoa with α- -fucosidase, α- -galactosidase and β-N-acetylhexosaminidase, but not with other glycosidases, trypsin and arylsulphatase, resulted in the complete inhibition of fertilization. Inhibitory saccharides and glycosidases did not interfere with sperm motility and had no effect on sperm-oolemma fusion. The pretreatment of cumulus-free oocytes with these agents did not inhibit sperm zona pellucida binding either. These results provide evidence that sperm-zona pellucida binding is mediated by ligands on the sperm surface containing fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine residues.     

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model. The quasi-steady state force on the microvillus at any given length was approximately fourfold lower in cells treated with cytochalasin D or when pulled with concanavalin A-coated rather than KPL-1-coated beads. These data suggest that associations between PSGL-1 and the underlying actin cytoskeleton significantly affect the early stages of leukocyte deformation under flow.     

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.     

Protein engineering by cell-surface display

Protein libraries displayed on cell surfaces can be labeled with soluble ligands exhibiting well-characterized binding equilibria and dissociation kinetics, and then quantitatively screened by flow cytometry at a rate of >10(4) clones/second. The promise of cell-surface display for directed evolution is being realized, with significant improvements recently reported in protein ligand binding affinity, stability, expression and enzymatic activity.