Low Binding Rate Research Articles

Ultrasensitive Photoelectrochemical Immunoassay Strategy Based on Bi2S3/Ag2S for the Detection of the Inflammation Marker Procalcitonin

As an inflammatory marker, procalcitonin (PCT) is more representative than other traditional inflammatory markers. In this work, a highly efficient photoelectrochemical (PEC) immunosensor was constructed based on the photoactive material Bi2S3/Ag2S to realize the sensitive detection of PCT. Bi2S3 was prepared by a hydrothermal method, and Ag2S quantum dots were deposited on the ITO/Bi2S3 surface via in situ reduction. Bi2S3 is a kind of admirable photoelectric semiconductor nanomaterial on account of its moderate bandgap width and low binding rate of photogenerated electron holes, which can effectively convert light energy into electrical energy. Therefore, based on the energy level matching principle of Bi2S3 and Ag2S, a labeled Bi2S3/Ag2S PEC immunosensor was constructed, and the sensitive detection of PCT was successfully established. The linear detection range of the PEC immunosensor was 0.50 pg∙mL-1 to 50 ng∙mL-1, and the minimum detection limit was 0.18 pg∙mL-1. Compared with the traditional PEC strategy, the proposed PEC immunosensor is simple, convenient, and has good anti-interference, sensitivity, and specificity, which could provide a meaningful theoretical basis and reference value for the clinical detection of PCT.

Antibody-Drug Conjugate to Treat Meningiomas.

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of <5% to the normal arachnoidal AC07 cells. The In Vivo Imaging System (IVIS) imaging demonstrated that the Cy5.5-labeled ADC targeted and accumulated in meningioma xenograft but not in normal organs. The pharmacokinetics study and histological analysis confirmed the stability and minimal toxicity. In vitro anti-cancer cytotoxicity indicated a high potency of ADC with an IC50 value of <10 nM. In vivo anti-tumor efficacy showed that the anti-SSTR2 ADC with doses of 8 and 16 mg/kg body weight effectively inhibited tumor growth. This study demonstrated that the anti-SSTR2 ADC can target meningioma and reduce the tumor growth.

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT.

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.

Investigating the Binding Mechanisms of Intrinsically Disordered Transactivation Domains to the TAZ1 Domain of CBP via Molecular Dynamics Simulation

Intrinsically disordered proteins play important functions in various biological processes. A number of intrinsically disordered transactivation domains (TADs) interact with the TAZ1 domain of the transcriptional coactivator CREB binding protein. In this work, we focus on five disordered TADs whose structures in complex with the TAZ1 domain have been determined via solution NMR. They are CITED2, HIF-1α, p53, STAT2, and RelA. Upon binding to TAZ1, these five TADs wrap around TAZ1 and undergo partial disorder-to-order transitions. A comparison of the TAD/TAZ1 complexes shows that the five TADs wrap around TAZ1 with different orientations and occupy the four TAZ1 binding sites with various orders. To elucidate the microscopic molecular details of the heterogeneous binding processes of TADs with TAZ1, we carried out molecular dynamics simulations using a coarse-grained topology-based model. Free energy analysis showed that intramolecular native contacts increased gradually with intermolecular native contacts for the five TADs. Φ-value analysis showed that only a small fraction of native contacts were formed in the transition state. Kinetics analysis revealed distinct features for the five TADs. HIF-1α and STAT2 bind to TAZ1 with low binding rates, slow transition kinetics, and low evolving probability. CITED2 and RelA bind to TAZ1 with high binding rates, medium transition kinetics, and high evolving probability. p53 binds to TAZ1 with high binding rates, high transition kinetics, and low evolving probability. As the five TADs have low sequence similarity and bind to TAZ1 with different binding poses, the similar and different binding features should reflect the roles that TADs and TAZ1 played in the binding process. Analysis of the binding mechanisms suggests that binding of TADs to TAZ1 is encoded by the TADs as well as templated by the TAZ1.

Investigation of erythrocyte membranes in exacerbation of chronic rhinosinusitis without nasal polyps by the method of fluorescent probes

Objective was to assess the state of lipid bilayer of human red blood cells in exacerbation of chronic rhinosinusitis without nasal polyps.Material and methods. Fluorescent probes O1O, O6O and РH7, which are ortho-hydroxy derivatives of oxazole, whose molecules can noncovalently bind to cell membranes and react to microenvironmental changes, were used in our experiment. Fluorescence of probes was determined in erythrocyte suspensions in patients with exacerbation of rhinosinusitis without nasal polyps and healthy individuals with deviated nasal septum who formed the control group.Results. The development of the pathological process investigated in our research is not accompanied by changes in the regions where O1O and O6O probes are located. However, exacerbation of chronic rhinosinusitis without nasal polyps is associated with a decrease in the number of molecules of the most hydrophobic РН7 probe bound to the membrane of erythrocytes during a one-hour incubation, which indicates a low binding rate of this probe to the membrane.Conclusions. There are no changes in the region of glycerol residues of phospholipids, in the region of carbonyl groups of phospholipids and in the region of fatty acid chains of phospholipids of erythrocyte membranes in patients with exacerbation of chronic rhinosinusitis without nasal polyps. The results obtained while using the most hydrophobic probe suggest that the development of inflammation is accompanied by an increase in hydration of the most polar region of erythrocyte membranes: the region of polar heads of phospholipids.

Identifying the Molecular Mechanics and Binding Dynamics Characteristics of Potent Inhibitors to HIV‐1 Protease

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the primary inhibition targets for chemotherapy of AIDS because of its critical role in the replication cycle of the HIV. In this work, a combinatory coarse-grained and atomistic simulation method was developed for dissecting molecular mechanisms and binding process of inhibitors to the active site of HIV-1 PR, in which 35 typical inhibitors were trialed. We found that the molecular size and stiffness of the inhibitors and the binding energy between the inhibitors and PR play important roles in regulating the binding process. Comparatively, the smaller and more flexible inhibitors have larger binding energy and higher binding rates; they even bind into PR without opening the flaps. In contrast, the larger and stiffer inhibitors have lower binding energy and lower binding rate, and their binding is subjected to the opening and gating of the PR flaps. Furthermore, the components of binding free energy were quantified and analyzed by their dependence on the molecular size, structures, and hydrogen bond networks of inhibitors. Our results also deduce significant dynamics descriptors for determining the quantitative structure and property relationship in potent drug ligands for HIV-1 PR inhibition.

Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: In vitro biological and binding activities.

e13030 Background: The monoclonal antibodies cetuximab (chimeric IgG1), necitumumab (human IgG1), and panitumumab (human IgG2) competitively inhibit ligand binding to epidermal growth factor receptor (EGFR) in domain 3. These antibodies block ligand induced EGFR activation and downstream molecular signaling. Beyond protein backbone differences, further differentiation of these EGFR antibodies is an important area of investigation. Methods: The effect on the in vitro proliferation of DiFi human colorectal cancer cells was assessed by ATP luminescence. The binding of the antibodies to the soluble extracellular domain of EGFR was evaluated by ELISA and surface plasmon resonance (SPR). Inhibition of ligand induced EGFR activation and downstream signaling was evaluated utilizing electrochemiluminescence method. Finally, binding kinetics was analyzed by SPR using a capture method. Results: The necitumumab concentration producing a half-maximal anti proliferative response (IC50) in DIFI cells was comparable to that of cetuximab. In contrast, a 1.6 fold greater concentration of panitumumab was required to produce the similar level of anti- proliferative response. This observation is supported by in vitro binding assays, which indicated that cetuximab and necitumumab binds to EGFR more effectively than panitumumab. Furthermore, the IC50 of nectitumab for the inhibition of EGFR phosphorylation was at least two fold greater than that of panitumumab. When EGFR binding kinetics were analyzed by SPR, panitumumab showed a much higher affinity (Kd) to EGFR than necitumumab and cetuximab, with a lower binding rate (kon) and a much slower dissociation rate (koff). Given the results in functional assays and the SPR data indicates that the high binding affinity (Kd) of panitumumab does not correlate with improved ability to block EGFR activation, downstream pathway activation and tumor cell proliferation. Conclusions: Although, as published, panitumumab has a higher affinity (Kd) than cetuximab and necitumumab, in vitro functional assays indicate that necitumumab and cetuximab have equal or greater ability to impact EGFR signaling and effect downstream activation of EGFR.

PRODUCTION OF ANTI-PMSG ANTIBODIES AND ITS RELATION TO THE PRODUCTIVITY OF RABBIT DOES

A batch of 100 female rabbits of the Hyplus breed were subjected to artificial insemination (A.I.) for a period of 9 months. With the goal of inducing the sexual receptivity of the does, half of them received systematic doses of 25 I.U .. of PMSG (Ciclogonine PROCHENA), 48 hours before A.I.. The other half were not injected (Control group). Following this experimental period (40 days after the last injection), blood samples of all the does were taken, in order to assay the anti-PMSG antibodies and to study the relationship between the production of antibodies, and the receptivity and productivity of the does observad for the previous litter. For the PMSG group, we classified the results of the does according to their level of immunity which either has a low binding rate (≤4 %) or a high one (> 4 %). 30,6% of the does from the group treated with PMSG produced anti­PMSG antibodies with a high binding rate. In no way related to the level of production of the antibodies, the PMSG treated does were found to be significantly more receptiva than the does in the control group (respectively 70.6 - 66.7 vs 42.0%, P = 0.023), while the fertility did not appear to have been either improved or decreased by the PMSG treatment. The total number of young born, born alive and weaned rabbits per litter did not vary according to the treatment or the intensity of antibody production. Likewise the weight of the litter and also the individual mean weight of the weaned young did not vary significantly according to the group. However, inside of the group treated with PMSG, the individual weight at weaning was the highest for the young from hyper- immune mothers (710 vs 639g, P = 0.035). For 29 does from the PMSG group, a second blood sample was taken 12 weeks after the previous one. Only 3 does showed a decreased level of antibodies, 4 months after the last injection. The study showed that one third of the does developed an immune reaction following injections of PMSG; however the proportion of antibodies produced did not appear related to either their sexual receptivity or productivity.

Simulation Study of the Contribution of Oligomer/Oligomer Binding to Capsid Assembly Kinetics

The process by which hundreds of identical capsid proteins self-assemble into icosahedral structures is complex and poorly understood. Establishing constraints on the assembly pathways is crucial to building reliable theoretical models. For example, it is currently an open question to what degree overall assembly kinetics are dominated by one or a few most efficient pathways versus the enormous number theoretically possible. The importance of this question, however, is often overlooked due to the difficulties of addressing it in either theoretical or experimental practice. We apply a computer model based on a discrete-event simulation method to evaluate the contributions of nondominant pathways to overall assembly kinetics. This is accomplished by comparing two possible assembly models: one allowing growth to proceed only by the accretion of individual assembly subunits and the other allowing the binding of sterically compatible assembly intermediates any sizes. Simulations show that the two models perform almost identically under low binding rate conditions, where growth is strongly nucleation-limited, but sharply diverge under conditions of higher association rates or coat protein concentrations. The results suggest the importance of identifying the actual binding pattern if one is to build reliable models of capsid assembly or other complex self-assembly processes.

Mechanism of Ca++ release from the sarcoplasmic reticulum: a computer model.

The proposed model describes myocyte calcium (Ca++) cycling, emphasizing the kinetics of sarcoplasmic reticulum (SR) Ca++ release channels. The suggested SR channel regulating mechanism includes two types of Ca++ binding sites: (1) low affinity sites with high binding rates, regulating the opening of Ca++ channels and (2) high affinity sites with low binding rates, which regulate their closing. The amount of Ca++ released from the SR and the peak value of Ca++ ion concentration [Ca++] in the cytoplasm were found to depend on the rate of the increase of [Ca++], similar to Ca++ induced Ca++ release experiments. The model describes spontaneous release of Ca++ from overloaded SR. The dependence of the control mechanism on the activating and inactivating sites is substantiated by simulations of ryanodine intervention, providing results similar to experimental results. Simulations under conditions of isolated SR vesicles produced Ca++ release results similar to measured data. Consequently, it is suggested that the recovery of Ca++ release channels represents the rate limiting factor in the process of mechanical restitution.

A Computer Model for Sarcoplasmic Reticulum Function In Ca++ Cycling in a Single Myocyte

The model of the sarcoplasmic reticulum (SR) calcium release mechanism is based on reported experimental data of Ca++ cycling that include kinetics of Ca++ release, interaction with Ca++ buffers in the myoplasm, and Ca++ uptake. The model accounts for the SR channels that affect the regulation of Ca++ release. The SR channel regulating mechanism includes positive and negative feedback loops associated in the model with two types of Ca++ binding sites: 1) low affinity sites with high binding rates, representing the activation process that enhance Ca++ release from the channel ana 2) high affinity sites with low binding rates, representing the inactivation process. Ca++ release regulation by SR channels gives a satisfactory description of the mechanical restitution behavior.

Two-dimensional crystallization of catalase on a monolayer film of poly(1-benzyl- l-histidine) spread at the air/water interface

Two-dimensional (2D) crystals of beef liver catalase were prepared by adsorption to a film of synthetic polypeptide, poly(1-benzyl- l-histidine) (PBLH), spread at the air/water interface. The crystallization experiments were carried out in the pH range of 4.8-6.4 for catalase solutions at low concentration (10 μg/ml). The pH-dependence suggested an electrostatic interaction in the binding of catalase to the PBLH film. At lower pH, small crystals were formed at a low binding rate, and at higher pH the binding was rapid and densely-packed 2D arrays with poor crystallinity were formed. To stimulate crystal growth, a thermal treatment was applied. One-shot heating of the interfacial catalase-PBLH film to 35–40°C was remarkably effective to form larger 2D crystals. The structure of catalase 2D crystals has been analyzed by Fourier filtering of the transmission electron micrographs. The crystal form is a new one, containing four catalase molecules in the unit cell with lattice parameters of a = 187 ○ A, b = 225 ○ A and γ = 92.8°.

Radiolabeled quaternary carbapenems and their interactions with human serum albumin.

A simple method is described for labeling carbapenems with [14C]dimethyl sulfate. Reverse-phase high-performance liquid chromatography of the reaction was used to purify the product. Carbapenems with 2-substituents containing pyrid-3-yl and -4-yl moieties could be labeled by this method, but those containing a pyrid-2-yl group could not. Nonreversible binding of these labeled carbapenems to human serum albumin was investigated. Pyridinium-3-yl compounds displayed low binding rates (0.028 to 0.044%/h), whereas three of four pyridinium-4-yl compounds bound much faster (0.38 to 0.62%/h). It is postulated that these differences are related to the ability of the compound to stabilize a deprotonated form transiently.

The reaction of aromatic peptides with double helical DNA. Quantitative characterisation of a two step reaction scheme.

The binding of LysTrpLys and LysTyrLys to calf thymus DNA has been investigated by the field jump method using fluorescence detection. Two separate relaxation processes, clearly distinguished on the time scale and by opposite amplitudes, are observed for the binding of LysTrpLys to DNA with approximately 30000 base pairs. The concentration dependence of the relaxation time constants demonstrates a mechanism with a bimolecular step followed by a slow intramolecular transition with a forward rate of 6.4 X 10(3) s-1 and an equilibrium constant of 11. Measurements at various degrees of peptide binding demonstrate that the binding mechanism associated with low binding rates is restricted to a rather low number of binding sites (roughly one site in 15 base pairs). The binding of LysTyrLys to the same DNA is not associated with relaxation processes of opposite amplitudes; nevertheless two processes could be identified and assigned to a two step mechanism corresponding to that observed in the case of LysTrpLys. In the presence of sonicated DNA both peptides show a single relaxation process with characteristics similar to those observed for the slow process in the binding to high molecular DNA. The data indicate that the intramolecular step is faster for low than for high molecular DNA. these results suggest an assignment of the intramolecular step to an insertion of the aromatic residues into the DNA associated with bending of the helix. The increase in the rate of the intramolecular step with decreasing chain length of the DNA may then be explained by a higher flexibility of the double helix at lower chain lengths.

Influence of ouabain on Ca exchangeability in resting atria of guinea-pig

The influence of ouabain (4×10 −7 mol) on total tissue calcium and calcium exchange of isolated resting atria was determined after different periods of exposure. The total tissue calcium content remained unchanged but the calcium exchange was, however, considerably accelerated in the presence of ouabain. The results suggest that the action of ouabain on the cellular calcium exchange also proceeds in resting muscles. The effect develops slowly and may be correlated to the low binding rate of ouabain under conditions of rest.