Kinetics of adhesion mediated by extracellular loops of claudin-2 as revealed by single-molecule force spectroscopy

Claudins (Cldns) comprise a large family of important transmembrane proteins that localize at tight junctions where they play a central role in regulating paracellular transportation of solutes across epithelia. However, molecular interactions occurring between the extracellular domains of these proteins are poorly understood. Here, using atomic force microscopy, the adhesion strength and kinetic properties of the homophilic interactions between the two extracellular loops of Cldn2 (C2E1or C2E2) and full-length Cldn2 were characterized at the level of single molecule. Results show that while the first extracellular loop is sufficient for Cldn2/Cldn2 trans-interaction, the second extracellular loop does not interact with the full-length Cldn2, with the first extracellular loop, or with itself. Furthermore, within the range of loading rates probed (10²-10⁴ pN/s), dissociation of Cldn2/Cldn2 and C2E1/C2E1 complexes follows a two-step energy barrier model. The difference in activation energy for the inner and outer barriers of Cldn2/Cldn2 and C2E1/C2E1 dissociation was found to be 0.26 and 1.66 k_BT, respectively. Comparison of adhesion kinetics further revealed that Cldn2/Cldn2 dissociates at a much faster rate than C2E1/C2E1, indicating that the second extracellular loop probably has an antagonistic effect on the kinetic stability of Cldn2-mediated interactions. These results provide an insight into the importance of the first extracellular loop in trans-interaction of Cldn2-mediated adhesion.     

Claudin-4 Deficiency Results in Urothelial Hyperplasia and Lethal Hydronephrosis

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4 ^−/− mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4 ^−/− mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4 ^−/− mice showed slightly yet significantly increased fractional excretion of Ca²⁺ and Cl⁻, suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4 ^−/− mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4 ^−/− mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.     

Claudin-3 and Claudin-5 Protein Folding and Assembly into the Tight Junction Are Controlled by Non-conserved Residues in the Transmembrane 3 (TM3) and Extracellular Loop 2 (ECL2) Segments

The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.     

Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k ₊=0.3–1.4 s⁻¹ and k _off=0.05–0.25 s⁻¹, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm²) raised k ₊ in a diffusion-dependent manner, leaving k _off relatively unchanged. Increasing the temperature (23–37 °C) raised both k ₊ and k _off, allowing calculation of the activation energy of the chemical reaction (around 20 k _B T). Increasing the compressive force on the cell during contact (up to 60 pN) raised k ₊ in a logarithmic manner, probably through an increase in the contact area, whereas k _off was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond. Received: 2 November 1999 / Revised version: 6 March 2000 / Accepted: 19 April 2000     

α-Synuclein aggregation variable temperature and variable pH kinetic data: A re-analysis using the Finke–Watzky 2-step model of nucleation and autocatalytic growth

The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k₁) and autocatalytic growth (A + B → 2B, rate constant k₂). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k₁) and growth (k₂) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG^‡, ΔH^‡, and ΔS^‡ are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔG^‡_nucleation = 23(3) kcal/mol; ΔG^‡_growth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k₁ value at pH ~ 3, as well as minimal k₁ near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k₂ values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k₁ vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k₁, nucleation step of α-synuclein. The chemically well-defined nucleation (k₁) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k₂ × [A]₀ (where k₂ is the rate constant for autocatalytic growth and [A]₀ is the initial protein concentration) is related to the empirical constant, k_app obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.     

Effects of Epidermal Growth Factor and Phorbol Ester on Thyroid Epithelial Integrity

The effects of epidermal growth factor (EGF) and phorbol ester (tetra-O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance (R_TE > 6 · 10³ Ω · cm²) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [³H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained (R_TE 1-3 · 10³ Ω · cm²; impermeability to [³H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca²⁺ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 μM) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [³H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca²⁺-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.     

Evidence against H+−HCO 3 − symport as the mechanism for HCO 3 − transport in the cyanobacteriumAnabaena variabilis

Summary The rate of inorganic carbon uptake and its steadystate accumulation ratio (intracellular/extracellular concentration) was determined in the cyanobacteriumAnabaena variabilis as a function of extracellular pH. The free energy of protons ( ) across the plasmalemma was calculated from determinations of membrane potential, and intracellular pH, as a function of the extracellular pH. While inward proton motive force decreased with increasing extracellular pH from 6.5 to 9.5, rate of HCO ₃ ^– influx and its accumulation ration increased. The latter is several times larger than would be expected should HCO ₃ ^– influx be driven by . It is concluded that HCO ₃ ^– transport in cyanobacteria is not driven by the proton motive force.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4.0–11.0) within the temperature range from 36 to 55^o. Solutions of CAT were exposed to LF (20.8 kHz) ultrasound (specific power, 48–62 W/cm²). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s^–1) of total inactivation (k _in), thermal inactivation (*k _in), and ultrasonic inactivation (k _in(us)). In all cases, the following inequality was valid: k _in > *k _in. The value of k _in(us) increased with the ultrasound power (range, 48–62 W/cm²) and exhibited a strong dependence on the pH of the medium. On increasing initial concentration of CAT (0.4–4.0 nM), k _in(us) decreased. The three rate constants were minimum within the range pH 6.5–8.0; their values increased considerably at pH < 6.0 and pH > 9.0. At 36–55^o, the temperature dependence of k _in(us) was characterized by an activation energy (E _act) of 19.7 kcal/mol, whereas the value of E _act for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 g/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (O ), prevented sonication-induced CAT inactivation at 10% (k _in and *k _in increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.     

Molecular recognition force spectroscopy study of the dynamic interaction between aptamer GBI‐10 and extracellular matrix protein tenascin‐C on human glioblastoma cell

Molecular recognition force spectroscopy (MR‐FS) was applied to investigate the dynamic interaction between aptamer GBI‐10 and tenascin‐C (TN‐C) on human glioblastoma cell surface at single‐molecule level. The unbinding force between aptamer GBI‐10 and TN‐C was 39 pN at the loading rate of 0.3 nN sec^?1. A series of kinetic parameters concerning interaction process such as the unbinding force f_u, the association rate constant k_on, dissociation rate constant at zero force k_off, and dissociation constant K_D for aptamer GBI‐10/TN‐C complexes were acquired. In addition, the interaction of aptamer GBI‐10 with TN‐C depended on the presence of Mg²⁺. This work demonstrates that MR‐FS can be used as an attractive tool for exploring the interaction forces and dynamic process of aptamer and ligand at the single‐molecule level. As a future perspective, MR‐FS may be used as a potential diagnostic and therapeutic tool by combining with other techniques. Copyright © 2012 John Wiley & Sons, Ltd.     

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (x _β), lifetimes in the absence of external perturbation (τ ⁰) and free energies (ΔG ^#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG ^# for these complexes. The values determined were up to 8 k_BT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Mechanisms of action of pH-induced effects on vascular smooth muscle

It is clear that pH has many effects on vascular smooth muscle and the overall action of pH on force will depend on the type of vascular smooth muscle in question and the combined effects on all the potential modulatory mechanisms. The major effects of pH on force appear to be mediated via modulation of [Ca]_i rather than changes in the sensitivity of the contractile machinery to Ca²⁺. There are still numerous gaps in our understanding of the actions of pH and as more data become available, we will be able to better understand the major mechanisms involved. (Mol Cell Biochem 263: 163–172, 2004)     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Efficient Inhibition of Collagen-Induced Platelet Activation and Adhesion by LAIR-2, a Soluble Ig-Like Receptor Family Member

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s⁻¹; IC₅₀ = 18 µg/ml) and high shear rate (1500 s⁻¹; IC₅₀ = 30 µg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and α2β1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis.     

Oxygen mass-transfer performance of low viscosity gas-liquid-solid system in a split-cylinder airlift bioreactor

The O₂ mass-transfer coefficient, k _L a, decreased by 20% when the viscosity of a simulated broth increased from 1.38 × 10^–3 to 3.43 × 10^–3 Pa s in a split-cylinder airlift bioreactor with a broth volume of 41 l. When the paper pulp concentration was below 10 g l^–1, k _L a hardly changed. While at 30 g l^–1, k _L a decreased by 56%. C₂O₄ ^2– and Na⁺ were found to have some effect on the k _L a value.     

Measuring kinetic dissociation/association constants between Lactococcus lactis bacteria and mucins using living cell probes

In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin–based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K_off). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K_on). Variations in the loading rate and contact time enabled us to determine K_on (3.3 × 10² M⁻¹·s⁻¹) and K_off (0.46 s⁻¹), and the latter was consistent with values given in the literature for sugar-protein interactions.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF₁₆₅ was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF₁₆₅ was dependent on the concentration of VEGF₁₆₅. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF₁₆₅ complexes was revealed, which was similar to that of the intact aptamer/VEGF₁₆₅. Besides, the dissociation rate constant (k_off) of split aptamer/VEGF₁₆₅ was close to that of intact aptamer/VEGF₁₆₅, and the interaction force of split aptamer/VEGF₁₆₅ was higher than the force of intact aptamer/VEGF₁₆₅. It indicated that split aptamer also possessed high affinity with VEGF₁₆₅. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.