Low Ligand Density Research Articles

The effects of ligand valency and density on the targeting ability of multivalent nanoparticles based on negatively charged chitosan nanoparticles

It has been shown that multivalent ligands could significantly enhance the binding avidity compared with the monovalent ones; therefore, once incorporated into nanoparticles, they promote superior targeting ability without increasing the ligand density. Although ligand valency and density play a key role on the targeting ability of corresponding nanoparticles, these facotrs remain largely unexplored and detailed studies are lacking. Herein, a series of multivalent ligands with certain valencies (FAn, n indicates the valency of ligand: n=3, 5, 7) has been conveniently synthesized by conjugating different copies of folate ligands with poly(acrylic acid) (PAA). Negatively charged chitosan nanoparticles (CTS-SA NPs) have been utilized as proper multivalent platforms because they can strongly suppress non-specific protein adsorption and cellular uptake without interfering with the targeting ability of multivalent ligands. Subsequently, the structure of CTS-SA NPs has been modified using different amounts of FAn to form multivalent nanoparticles (FAn-CTS-SA NPs) with various valencies and densities. A series of specific investigations of them suggested that the cellular uptake of multivalent nanoparticles has largely varied with the ligand valency variation even at similar ligand densities; and also largely varied with ligand density variation even at the same ligand valencies. The intermediate valency and density values determined in the current study (ie., 5 and 2.4wt%, respectively) have provided the best cellular uptake, facilitating superior targeting ability at relatively low ligand valency and density. Unexpectedly, no conspicuous difference has been observed during endocytotic inhibition assays with single inhibitors, which may be attributed to the synergetic endocytotic mechanism with multiple pathways of multivalent nanoparticles. The optimal multivalent nanoparticles have also exhibited excellent biocompatibility, long-term stability in vitro and enhanced circulation time in vivo, thus demonstrating their potential for targeted drug delivery.

Free energy analysis of cell spreading

In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to “V-shaped” and “Y-shaped” ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing cytoskeletal free energy and increasing passive elastic free energy.

In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates.

Unnatural sugar-mediated metabolic labeling of cancer cells, coupled with efficient Click chemistry, has shown great potential for in vivo imaging and cancer targeting. Thus far, chemical labeling of cancer cells has been limited to the small-sized azido groups, with the large-sized and highly hydrophobic dibenzocyclooctyne (DBCO) being correspondingly used as the targeting ligand. However, surface modification of nanomedicines with DBCO groups often suffers from low ligand density, difficult functionalization, and impaired physiochemical properties. Here we report the development of DBCO-bearing unnatural sugars that could directly label LS174T colon cancer cells with DBCO groups and subsequently mediate cancer-targeted delivery of azido-modified silica nanoconjugates with easy functionalization and high azido density in vitro and in vivo. This study, for the first time, demonstrates the feasibility of metabolic labeling of cancer cells with large-sized DBCO groups for subsequent, efficient targeting of azido-modified nanomedicines.

Silica-based strong anion exchange media for protein purification

The main objective of our research was to develop silica-based, polymer-functionalized ion exchange materials for single-use bioprocess applications, with the ultimate goal of achieving maximal binding capacity for target proteins. Herein we report the utilization of Grace® wide pore silica gel and bonding the silica with cationic polymers. The strong anion exchange materials have been prepared by a two-step process involving initial bonding with two trimethoxysilanes and subsequent aqueous solution radical polymerization with quaternary ammonium ion containing monomers and an azo initiator. Using the binding capacities for bovine serum albumin (BSA), a model protein for the evaluation of the new materials, we optimized the processes with regard to the median pore size of the silica gel, as well as polymer composition and ratios, which were determined by reagent ratios and reaction conditions. The products were also characterized by both chemical and physical methods. It has been found that higher binding capacities are associated with lower ligand density and higher molecular weight for the attached polymers, with over 20% higher in both static and dynamic binding capacity values with the same amount of attached polymers. The advantages of a large pore size distribution and optimal median pore size for the base silica are discussed. Optimal pore size range of 500–1500Å and distribution of Span 90 for over 1.0 give the highest BSA binding capacities. Silica-based strong anion exchange materials showed excellent flow characteristics when packed into a column and were superior to commercial agarose-based strong anion exchange material with respect to dynamic binding capacity, elution of proteins, and baseline separation of a mixture of three model proteins.

Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and pore sizes

Hydrophobic charge-induction chromatography (HCIC) is a novel technology for antibody purification. The ligand densities and pore properties of HCIC resins have significant effects on the separation behavior of protein, however, the understandings are quite limited. In the present work, new HCIC ligand, 2-mercapto-1-methylimidazole (MMI) was coupled to three agarose matrices with different pore sizes. A series of MMI resins with different ligand density and pore size was prepared by the control of ligand coupling. The adsorption isotherms and kinetics on the series of MMI resins were investigated with bovine serum immunoglobulin as the model IgG, and the effects of salt addition were studied. The Langmuir equation and pore diffusion model were used to fit the experimental data, and the influences of ligand density, pore size and salt addition on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. It was found that the adsorption capacities and the effective pore diffusion coefficient increased with the increase of ligand density and pore size. The effects of salt addition on the adsorption behaviors were dependent on the ligand density. For low ligand density the IgG adsorption was salt-promoted, while the resins with high ligand density showed a salt-independent property. The results indicated that for a given protein the ligand density and pore size of HCIC resins should be optimized for improving the protein adsorption.

Synthesis, characterisation and chromatographic evaluation of pentafluorophenyl and phenyl bonded silica phases prepared using supercritical carbon dioxide as a reaction solvent

Pentafluorophenyl and phenyl silica stationary phases offer alternative selectivity compared to alkyl bonded C18 and C8 stationary phases, through other interactions such as π–π interactions, dipole–dipole and hydrogen bond interactions. Pentafluorophenyl and phenyl silica bonded stationary phases were efficiently prepared in sc-CO2 specifically pentafluorophenyl propyl (PFPP), pentafluorophenyl (PFP), phenyl propyl (PP) and phenyl (P) silica stationary phases. The bonded phases were characterised by elemental analysis, thermogravimetric analysis (TGA), BET, and by solid-state NMR spectroscopy. Chromatographic performance of the supercritical fluid generated phases was also investigated using the Neue test. The authors present results which demonstrate that pentafluorophenyl and phenyl stationary phases can be prepared successfully under supercritical conditions of 100°C, 414bar in a reaction time of 1h with surface coverage comparable to traditional organic solvent based methods. Chromatographic results reveal that the pentafluorophenyl propyl (PFPP) phase provides superior separation performance for Neue test solutes despite having a lower ligand density (C: 5.67%, 2.2μmol/m2) compared to the phenyl propyl (PP) analogue having the highest ligand density (C: 6.67%, 2.5μmol/m2). The difference chromatographic performance is attributed to the polarity of the CF bond in PFPP phase. Moreover, as the alkyl chain length decreases, the hydrophobic interaction also decreases, and the PFPP phase (with a propyl linkage) provides better separation compared to the PFP phase.

Antigenic ligand density modulates execution of naïve CD8+ T cell response programme (110.1)

Abstract Upon brief exposure to antigenic stimulation, naïve CD8+ T lymphocytes execute an autonomous programme of several rounds of proliferation and maturation into effector cytotoxic cells. We have studied the role of ligand density as a modifier of this program using T cell receptor (TCR)-transgenic CD8+ T cell activation by short-duration stimulation in vitro. Over the entire ligand density range studied, we observe a graded increase in the frequency of responder naïve T cells that upregulate the expression of CD69, an early activation marker. However, the levels of induced CD69 are similar at all ligand densities. When CD69+ T cells are purified at early stages and cultured, most undergo successive proliferation cycles, showing that the execution of the autonomous programme during CD8+ T cell response is independent of ligand density. However, at very low ligand densities, the efficiency with which CD69+ T cells enter proliferation is poor and slow, leading to lower cell yields. At these ligand densities, post-activation TCR internalization is also inefficient. At any given ligand density, if activated CD69+ T cells that have internalised TCR are separated from those that have not internalised TCR, TCR-high cells show a lag in proliferation regardless of the ligand density. Together, these data indicate that ligand density quantitatively modulates the lag phase of the autonomous proliferation-differentiation programme of CD8+ T cells via TCR internalization-dependent mechanism.

Effect of Octreotide–Polyethylene Glycol(100) Monostearate Modification on the Pharmacokinetics and Cellular Uptake of Nanostructured Lipid Carrier Loaded with Hydroxycamptothecine

A new conjugate, octreotide-polyethylene glycol(100) monostearate (OPMS), was developed for the enhancement of targeting delivery of hydroxycamptothecine (HCPT) loaded in nanostructured lipid carrier (NLC). 2 × 10(-3) and 5 × 10(-3) mmol of OPMS were respectively used to modify NLC so that the targeted nanocarriers with low and high ligand density were obtained. For comparison, the pegylated NLCs without octreotide were prepared by adding equal molar amounts of polyethylene glycol(100) monostearate (PGMS). The relation between the modification levels and properties of various NLCs were studied in vivo and in vitro. At a high modification level, a slower release rate of HCPT and the more stable nanocarriers was achieved. At the same time, the fixed aqueous layer thickness (FALT) and average surface density of PEG chains (SD(PEG)) was increased, but the distance (D) between two neighboring PEG grafting sites became narrower. The in vivo pharmacokinetic study in healthy rat indicated that the modified NLCs had a longer circulation than NLC (P < 0.05) due to pegylation effect and OPMS modified NLCs had larger MRT and AUC(0-t) than that of PGMS modified NLCs at the same modification level. Furthermore, the florescence microscopy observation also showed the targeting effect of octreotide modification on somatostatin receptors (SSTRs) of tumor cell (SMMC-7721). The uptake of SMMC-7721 was much more than that of normal liver cell (L02) for OPMS modified NLC, and the highest uptake was observed for 5 × 10(-3) mmol of OPMS modified one. No obvious difference was found among the L02 uptake of OPMS modified NLCs and NLC, but their uptake was higher than that of PGMS modified NLCs. All the results indicated that the OPMS highly modified NLCs would improve the effect of antitumor therapy by inhibiting the degradation, evading RES and enhancing the drug uptake of tumor cells.

Quantitative analysis of multivalent ligand presentation on gold glyconanoparticles and the impact on lectin binding.

Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide an excellent platform for sensitive protein recognition. Using nanomaterials as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, the quantitative analysis of the binding affinity of these glyconanomaterials has been lacking. In this Article, we report a new method to measure the binding affinity of glyconanoparticle (GNP)-protein interactions based on a fluorescent competition binding assay, which yielded the apparent dissociation constant (K(d)) of GNPs with the interacting protein. Au nanoparticles conjugated with underivatized mono-, oligo-, and polysaccharides were synthesized using our recently developed photocoupling chemistry. The affinities of these GNPs with lectins were measured and were several orders of magnitude higher than the corresponding free ligands with lectins. The effect of ligand display on the binding affinity of GNPs was, furthermore, studied where GNPs of varying linker type, spacer length, ligand density, and nanoparticle size were prepared and K(d) values determined. The long spacer linker containing hydrocarbon and ethylene oxide units gave the highest binding affinity as well as assay sensitivity. The binding affinity increased with ligand density in general, showing a drastic increase in affinity at low ligand density. In addition, the affinity enhancement was more pronounced on smaller NPs than the larger ones. These results not only demonstrate that the binding affinity of GNPs is highly influenced by how the ligands are presented on the nanoparticles but also pave the way for tailor-made glyconanomaterials with tunable affinity by way of ligand display.

Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone

Substrates with mechanical property gradients and various extracellular matrix ligand loadings were evaluated for their ability to direct bone marrow stromal cell differentiation along osteogenic and tenogenic lineages. After verifying reproducible mechanical compliance characteristics of commercial hydrogel gradient substrates, substrates were functionalized with whole length fibronectin or collagen, both of which are found in skeletal structures and are relevant to cell-matrix signalling. Bone marrow stromal cells were seeded onto the substrates in growth media and cultured first to examine cell attachment and morphology, indicating higher levels of attachment on collagen substrates after 1 h, and increased spreading and organization trends after 24 h. Differentiation studies showed an increase in osteoblast differentiation on fibronectin substrates while collagen substrates lacked osteogenic differentiation. Osteogenic differentiation decreased on substrates of lower stiffness and lower ligand density. Molecular investigations revealed an increase in relevant signalling molecules for osteoblasts that were consistent with differentiation studies, but detected the presence of tenoblast markers on collagen substrates within a narrow range of stiffness. Our results indicate that mechanovariant substrates do hold promise as a culture platform for directed differentiation to tendon and bone by altering gene level expression of relevant signalling molecules. This study aids in understanding the molecular mechanisms that drive differentiation from substrate based cues, and could aid the design of therapeutic biomaterials at the transition from tendon to bone.

Glyconanomaterials: synthesis, characterization, and ligand presentation.

Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins.

3D structure-based protein retention prediction for ion-exchange chromatography

The interest in understanding fundamental mechanisms underlying chromatography drastically increased over the past decades resulting in a whole variety of mostly semi-empirical models describing protein retention. Experimental data about the molecular adsorption mechanisms of lysozyme on different chromatographic ion-exchange materials were used to develop a mechanistical model for the adsorption of lysozyme onto a SP Sepharose FF surface based on molecular dynamic simulations (temperature controlled NVT simulations) with the Amber software package using a force-field based approach with a continuum solvent model. The ligand spacing of the adsorbent surface was varied between 10 and 20A. With a 10A spacing it was possible to predict the elution order of lysozyme at different pH and to confirm in silico the pH-dependent orientation of lysozyme towards the surface that was reported earlier. The energies of adsorption at different pH values were correlated with isocratic and linear gradient elution experiments and this correlation was used to predict the retention volume of ribonuclease A in the same experimental setup only based on its 3D structure properties. The study presents a strong indication for the validity of the assumption, that the ligand density of the surface is one of the key parameters with regard to the selectivity of the adsorbent, suggesting that a high ligand density leads to a specific interaction with certain binding sites on the protein surface, while at low ligand densities the net charge of the protein is more important than the actual charge distribution.

Integrin Clustering Is Driven by Mechanical Resistance from the Glycocalyx and the Substrate

Integrins have emerged as key sensory molecules that translate chemical and physical cues from the extracellular matrix (ECM) into biochemical signals that regulate cell behavior. Integrins function by clustering into adhesion plaques, but the molecular mechanisms that drive integrin clustering in response to interaction with the ECM remain unclear. To explore how deformations in the cell-ECM interface influence integrin clustering, we developed a spatial-temporal simulation that integrates the micro-mechanics of the cell, glycocalyx, and ECM with a simple chemical model of integrin activation and ligand interaction. Due to mechanical coupling, we find that integrin-ligand interactions are highly cooperative, and this cooperativity is sufficient to drive integrin clustering even in the absence of cytoskeletal crosslinking or homotypic integrin-integrin interactions. The glycocalyx largely mediates this cooperativity and hence may be a key regulator of integrin function. Remarkably, integrin clustering in the model is naturally responsive to the chemical and physical properties of the ECM, including ligand density, matrix rigidity, and the chemical affinity of ligand for receptor. Consistent with experimental observations, we find that integrin clustering is robust on rigid substrates with high ligand density, but is impaired on substrates that are highly compliant or have low ligand density. We thus demonstrate how integrins themselves could function as sensory molecules that begin sensing matrix properties even before large multi-molecular adhesion complexes are assembled.

Integrin ανβ3 Targeted Gene Delivery Using RGD Peptidomimetic Conjugates with Copolymers of PEGylated Poly(ethylene imine)

This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.

Traction Stresses and Translational Distortion of the Nucleus During Fibroblast Migration on a Physiologically Relevant ECM Mimic

Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.

Optical Tweezers and Fluorescence Recovery After Photo-Bleaching to Measure Molecular Interactions at the Cell Surface

This paper describes applications of two optical microscopy techniques, namely laser tweezers and fluorescence recovery after photo-bleaching (FRAP), to the measurement of ligand/receptor/cytoskeleton interactions. These methods are used in combination with ligand-coated microspheres, binding to specific membrane receptors at the dorsal cell surface. A first application exploits the possibility to impose piconewton forces on microspheres. At low ligand density, one can identify the rupture of individual bonds between ligand/receptor complexes and the motile actin cytoskeleton. The second application uses control of the initial contact time by optical tweezers, together with time lapse imaging of GFP-tagged receptor accumulation around the microspheres. Quantification of fluorescence enrichment together with a simple chemical reaction model allows characterization of global ligand/receptor on-rates. These contacts eventually reach steady-state corresponding to continuous formation and dissociation of ligand/receptor bonds. FRAP is then used to probe the equilibrium dynamics of this system, by photo-bleaching GFP-tagged receptors recruited at ligand-coated beads. The recovery curves are fitted by a diffusion/reaction model, to yield turnover rates between adhesion proteins. To illustrate the potential of these techniques, we take examples from our previously published data on neuronal adhesion proteins involved in growth cone locomotion, in particular N-cadherin and immunoglobulin cell adhesion molecules.

DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

DNA-induced aggregation and contraction of expanded bed adsorption chromatography beds have been examined using strong anion exchanger Q HyperZ and calf thymus DNA in buffers containing added NaCl. Two batches of adsorbent with different ionic capacities were used allowing the effects of different ligand densities to be examined. Very high dynamic binding capacities at 10% breakthrough were found in the absence of added salt. However, the highest binding capacities (∼10 and ∼19 mg DNA ml −1 gel) were found in buffers containing added salt at concentrations of either 0.25 or 0.35 M, for the low and high ligand density adsorbents, respectively. Bed contraction was observed, but did not correlate with dynamic binding capacity or with the amount of DNA loaded. No differences in bed contraction were seen by varying the concentration of DNA loaded in the range of 20–80 μg ml −1 even though the dynamic binding capacity was reduced as DNA concentration was increased. The extent of bed contraction during DNA loading was found to be a function of added salt concentration and ligand density of the adsorbent. The results imply that ligand density significantly affects the salt tolerance of anion exchangers when binding DNA. However, more importantly, with the adsorbents examined here, attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture in real feedstocks.

T-Cell Activation: A Queuing Theory Analysis at Low Agonist Density

We analyze a simple linear triggering model of the T-cell receptor (TCR) within the framework of queuing theory, in which TCRs enter the queue upon full activation and exit by downregulation. We fit our model to four experimentally characterized threshold activation criteria and analyze their specificity and sensitivity: the initial calcium spike, cytotoxicity, immunological synapse formation, and cytokine secretion. Specificity characteristics improve as the time window for detection increases, saturating for time periods on the timescale of downregulation; thus, the calcium spike (30s) has low specificity but a sensitivity to single-peptide MHC ligands, while the cytokine threshold (1h) can distinguish ligands with a 30% variation in the complex lifetime. However, a robustness analysis shows that these properties are degraded when the queue parameters are subject to variation—for example, under stochasticity in the ligand number in the cell-cell interface and population variation in the cellular threshold. A time integration of the queue over a period of hours is shown to be able to control parameter noise efficiently for realistic parameter values when integrated over sufficiently long time periods (hours), the discrimination characteristics being determined by the TCR signal cascade kinetics (a kinetic proofreading scheme). Therefore, through a combination of thresholds and signal integration, a T cell can be responsive to low ligand density and specific to agonist quality. We suggest that multiple threshold mechanisms are employed to establish the conditions for efficient signal integration, i.e., coordinate the formation of a stable contact interface.

Improving recovery of recombinant hepatitis B virus surface antigen by ion-exchange chromatographic supports with low ligand density

Ion-exchange chromatography (IEC) with the lowest step recovery (<50%) is still a bottleneck of the essentially four-step chromatographic procedure for the purification of recombinant hepatitis B surface antigen (r-HBsAg) from transformed Chinese hamster ovary (CHO) cell line. The low recovery is mainly resulted from the change of the assemble structure of HBsAg particles such as disassociation or aggregation on the surface of the absorbent during binding and eluting procedures. To improve the chromatographic efficiency of IEC, the influence of ligand density from 0.041 to 0.130 mmol diethylaminoethyl (DEAE)/ml absorbent on IEC of r-HBsAg was investigated. The retention time of HBsAg particles depended highly on ligand density. Higher HBsAg recovery and purification factor (PF) were obtained from the absorbents with lower ligand density. Under the optimum chromatographic conditions, the HBsAg recovery was increased from about 44% to 66% and the PF was increased from 9 to 13 by decreasing the ligand density from 0.130 to 0.041 mmol DEAE/ml absorbent.

The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure

The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of one of the basic TM residues of NKG2D resulted in loss of two DAP10 chains, indicating that each TM arginine serves as an interaction site for a DAP10 dimer. Assembly of the hexameric structure was cooperative because this mutation also significantly reduced NKG2D dimerization. A monomeric NKG2D TM peptide was sufficient for assembly with a DAP10 dimer, indicating that the interaction between these proteins occurs in the membrane environment. Formation of a three-helix interface among the TM domains involved ionizable residues from all three chains, the TM arginine of NKG2D and both TM aspartic acids of the DAP10 dimer. The organization of the TM domains thus shows similarities to the T cell antigen receptor-CD3 complex, in particular to the six-chain assembly intermediate between T cell antigen receptor and the CD3delta epsilon and CD3gamma epsilon dimers. Binding of a single ligand can thus result in phosphorylation of four DAP10 chains, which may be relevant for the sensitivity of NKG2D receptor signaling, in particular in situations of low ligand density.