Low-affinity Binding Sites Research Articles

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ− subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ− site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.

Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α1,2,3,5β2γ2S GABAARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABAAR than at the α1,2,3,5β2γ2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation exerted by clobazam, N-desmethylclobazam and clonazepam at the α1β2γ2S, α2β2γ2S, α3β2γ2S and α5β2γ2S GABAARs indicate that the observed clinical differences between clobazam and 1,4-benzodiazepines are likely to arise from factors other than their respective pharmacological properties at the GABAARs as investigated here.

Increased Sensitivity to Cocaine Self-Administration in HIV-1 Transgenic Rats is Associated with Changes in Striatal Dopamine Transporter Binding.

Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [(125)I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125)I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the ascending limb of the cocaine dose-response curve observed in HIV-1 Tg vs. F344 rats, and has major implications for the function of cocaine binding to DAT in HIV patients. The absence of HIV-related changes in heroin intake are likely due to less dopaminergic involvement in the mediation of heroin reward, further emphasizing the preferential influence of HIV on dopamine-related behaviors.

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-links cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.

Comparing foam and interfacial properties of similarly charged protein–surfactant mixtures

The foam stability of protein–surfactant mixtures strongly depends on the charge of the protein and the surfactant, as well as on their mixing ratio. Depending on the conditions, the mixtures will contain free proteins, free surfactants and/or protein–surfactant complexes. To be able to compare different protein–surfactant mixtures, generic knowledge about the occurrence of each of these states and their relative contribution to foam stability is essential. In this work, the foam stability and interfacial properties of bovine serum albumin (BSA) mixed with sodium dodecyl sulphate (SDS) as well the binding of SDS to BSA as are studied at different molar ratios (MR). A comparison is made with β-lactoglobulin (BLG) mixed with SDS. Both proteins and SDS are negatively charged at pH 7. The foam stability in the presence of small amounts (up to MR 1) of SDS is half the value of the pure protein solutions. The foam stability for both protein surfactant mixtures reaches a minimum at MR 20. A further increase of the MR leads to an increase of foam stability. The foam stability of BLG–SDS at MR >20 follows the foam stability of pure SDS solutions at equivalent concentrations, while BSA–SDS mixtures have an offset and begin to increase from MR >50. This behaviour was also reflected in the surface pressure and complex dilatational elastic moduli, and could be linked to the binding of the surfactant to the proteins. Both proteins bind SDS at high and low affinity binding sites. BSA's high affinity binding sites have a binding stoichiometry of 5.5 molSDS/molprotein, and BLG's high affinity binding site has a stoichiometry of 0.8 molSDS/molprotein (determined by isothermal titration calorimetry). Binding to the low affinity binding sites, occurs with a binding ratio, leading to an accumulation of free surfactants. While the basic mechanisms underlying the foam properties of mixed systems are not explained in detail by this approach, the foam stability plots of both protein surfactant mixtures could be superimposed using the concentration of free SDS.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV–VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill’s coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

Impact of viral activators and epigenetic regulators on HIV-1 LTRs containing naturally occurring single nucleotide polymorphisms.

Following human immunodeficiency virus type 1 (HIV-1) integration into host cell DNA, the viral promoter can become transcriptionally silent in the absence of appropriate signals and factors. HIV-1 gene expression is dependent on regulatory elements contained within the long terminal repeat (LTR) that drive the synthesis of viral RNAs and proteins through interaction with multiple host and viral factors. Previous studies identified single nucleotide polymorphisms (SNPs) within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3T, C-to-T change at position 3, and 5T, C-to-T change at position 5 of the binding site, respectively, when compared to the consensus B sequence) that are low affinity binding sites and correlate with more advanced stages of HIV-1 disease. Stably transfected cell lines containing the wild type, 3T, 5T, and 3T5T LTRs were developed utilizing bone marrow progenitor, T, and monocytic cell lines to explore the LTR phenotypes associated with these genotypic changes from an integrated chromatin-based microenvironment. Results suggest that in nonexpressing cell clones LTR-driven gene expression occurs in a SNP-specific manner in response to LTR activation or treatment with trichostatin A treatment, indicating a possible cell type and SNP-specific mechanism behind the epigenetic control of LTR activation.

Interactions of Cell Surface Glycoproteins

Cell surfaces are coated with glycoproteins and glycolipids. It is shown that carbohydrates present on cell surfaces offer a potential low affinity binding sites that act as diverse carbohydrate-carbohydrate recognition systems. Weak interactions between carbohydrate molecules allow the cells to test surrounding surfaces prior to establishing more stable covalent bonds. Carbohydrate-Carbohydrate interactions on cell surfaces play important roles in various biological processes such as cell trafficking, host-pathogen interactions, embryogenesis, spermatogenesis, implantation, cancer progression and angiogenesis. To further analyze the interactions between carbohydrates in biological systems, we are investigating the force-spectroscopy of mannobiosylated BSA on coated AFM surfaces. We present the effect of the oligosaccharide conformation and salt dependence on the glycoprotein interactions.

Low Affinity Binding Site Clusters Confer Hox Specificity and Regulatory Robustness

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity invitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding invivo, but multiple clustered sites were required for robust expression when embryos developed invariable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression.

Membrane-Type 1 Matrix Metalloproteinase Downregulates Fibroblast Growth Factor-2 Binding to the Cell Surface and Intracellular Signaling.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non-proteolytic interactions with extracellular, intracellular, and transmembrane proteins. Here we show that in tumor cells MT1-MMP downregulates fibroblast growth factor-2 (FGF-2) signaling by reducing the amount of FGF-2 bound to the cell surface with high and low affinity. FGF-2 induces weaker activation of ERK1/2 MAP kinase in MT1-MMP expressing cells than in cells devoid of MT1-MMP. This effect is abolished in cells that express proteolytically inactive MT1-MMP but persists in cells expressing MT1-MMP mutants devoid of hemopexin-like or cytoplasmic domain, showing that FGF-2 signaling is downregulated by MT1-MMP proteolytic activity. MT1-MMP expression results in downregulation of FGFR-1 and -4, and in decreased amount of cell surface-associated FGF-2. In addition, MT1-MMP strongly reduces the amount of FGF-2 bound to the cell surface with low affinity. Because FGF-2 association with low-affinity binding sites is a prerequisite for binding to its high-affinity receptors, downregulation of low-affinity binding to the cell surface results in decreased FGF-2 signaling. Consistent with this conclusion, FGF-2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1- MMP than in MT1-MMP expressing cells. Thus, MT1-MMP controls FGF-2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF-2.

Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur.The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman’s reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200–250nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue.Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was investigated by spectrofluorescence, ITC and 1H NMR spectroscopy, respectively. SSZ and SLD change the affinity of each other to the binding site in non- and modified human serum albumin. The presence of SLD causes the increase of association constant (Ka) of SSZ–oHSA system and the strength of binding and the stability of the complexes has been observed while in the presence of SSZ a displacement of SLD from the SLD–HSA has been recorded. The analysis of 1H NMR spectral parameters i.e. changes of chemical shifts of the drug indicate that the presence of SSZ and SLD have a mutual influence on changes in the affinity of human serum albumin binding site and this competition takes place not only due to the additional drug but also to the oxidation of HSA.

Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site.

Kinetic models for receptor-catalyzed conversion of coxsackievirus B3 to A-particles.

The immunoglobulin superfamily protein receptors for poliovirus, human rhinovirus, and coxsackievirus B (CVB) serve to bind the viruses to target cells and to facilitate the release of the virus genome by catalyzing the transition from the mature infectious virus to the A-particle uncoating intermediate. Receptor binding sites characterized by two equilibrium dissociation constants have been identified. The site with higher affinity is best observed at warmer temperatures and appears to correlate with the reversible conformational state in which the capsid is permeable to small molecules and peptides that are buried in the crystal structures are exposed. Measurements of CVB conversion to inactive particles over time in the presence of varied concentrations of soluble coxsackievirus and adenovirus receptor showed that the observed first-order rate constant varies with receptor concentration. The dose-response data, previously modeled as the sum of first-order reactions, have been used to evaluate models for the receptor-catalyzed conversion of CVB that include the high- and low-affinity binding sites associated with capsid breathing. Allosteric models wherein receptor binding shifts the equilibrium toward the open capsid conformation, in which the high-affinity binding site is available, best fit the data. This paper compares models that relate the structural, mechanistic, and kinetic details of receptor-virus interactions known from previous work with human enteroviruses. New models are derived using recent results from receptor-catalyzed conversion of coxsackievirus B3 to non-infectious A-particles. Of those considered, the acceptable models include the capsid breathing cycle and two conformation-dependent receptor binding sites. The results indicate that the receptor enhancement of virus conversion to A-particles involves allostery through conformation selection.

Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters.

ExsA is a member of the AraC/XylS family of transcriptional regulators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). All P. aeruginosa T3SS promoters contain two adjacent binding sites for monomeric ExsA. The amino-terminal domain of ExsA (NTD) is thought to mediate interactions between the ExsA monomers bound to each site. Threading the NTD onto the AraC backbone revealed an α-helix that likely serves as the primary determinant for dimerization. In this study, we performed alanine scanning mutagenesis of the ExsA α-helix (residues 136 to 152) to identify determinants required for self-association. Residues L137, C139, L140, K141, and L148 exhibited self-association defects and were required for maximal activation by ExsA. Disruption of self-association resulted in decreased binding to T3SS promoters, particularly loss of binding by the second ExsA monomer. Removing the NTD or increasing the space between the ExsA-binding sites restored the ability of the second ExsA monomer to bind the PexsC promoter. This finding indicated that, in the absence of self-association, the NTD prevents binding by a second monomer. Similar findings were seen with the PexoT promoter; however, binding of the second ExsA monomer in the absence of self-association also required the presence of a high-affinity site 2. Based on these data, ExsA self-association is necessary to overcome inhibition by the NTD and to compensate for low-affinity binding sites, thereby allowing for full occupation and activation of ExsA-dependent promoters. Therefore, ExsA self-association is indispensable and provides an attractive target for antivirulence therapies.

Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction.

The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 μM dermcidin, the platelets became resistant to the inhibitory effect of aspirin similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin antibody restored the sensitivity of the platelets to the aspirin effect. Incubation of AMI PRP pretreated with 15 μM aspirin, a stimulator of the NO synthesis, resulted in the increased production of NO in the platelets that removed the bound dermcidin by 40% from the high affinity binding sites of AMI platelets. When the same AMI PRP was retreated with 10 μM aspirin, the aggregation of platelets was completely inhibited by NO synthesis.

Soluble phosphatidylserine binds to two sites on human factor IXa in a Ca2+ dependent fashion to specifically regulate structure and activity.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

Preparation and Adsorption Characteristics of Cordycepin Molecularly Imprinted Polymers

The separation of a molecularly imprinted polymer for cordycepin was investigated. The synthesis employed cordycepin as the molecular template, alpha-methylacrylic acid as the functional monomer, glycol dimethyl acrylate as the cross-linking agent, azobisisobutyronitrile as the initiator, and tetrahydrofuran as the solvent and pore-foaming agent. The interaction between cordycepin and the functional monomer was investigated by ultraviolet-visible and infrared spectroscopy. The properties of the molecularly imprinted polymer were analyzed by scanning electron microscopy, equilibrium adsorption experiments, and the Scatchard equation. Static adsorption, solid phase extraction, and high-performance liquid chromatography experiments were employed to evaluate the adsorption properties and selective recognition characteristics. The results showed that the molecularly imprinted polymer had specific adsorption with cordycepin, and the maximum absorption capacity was 1920 µg/g. Scatchard analysis suggested that high affinity and low affinity binding sites were present. For the high affinity case, the dissociation constant and apparent maximum numbers of the binding sites were 0.0089 mmol/L and 4.78 µmol/g, respectively. The dissociation constant and apparent numbers of binding sites were 0.035 mmol/L and 6.047 µmol/g for the low affinity sites. Compared with the corresponding nonimprinted polymer, the cordycepin molecularly imprinted polymer exhibited higher adsorption and selectivity for cordycepin than structural analogs.

Changes in dopamine transporter binding in nucleus accumbens following chronic self-administration cocaine: heroin combinations.

Concurrent use of cocaine and heroin (speedball) has been shown to exert synergistic effects on dopamine neurotransmission in the nucleus accumbens (NAc), as observed by significant increases in extracellular dopamine levels and compensatory elevations in the maximal reuptake rate of dopamine. The present studies were undertaken to determine whether chronic self-administration of cocaine, heroin or a combination of cocaine:heroin led to compensatory changes in the abundance and/or affinity of high- and low-affinity DAT binding sites. Saturation binding of the cocaine analog [(125) I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125) I]RTI-55) in rat NAc membranes resulted in binding curves that were best fit to two-site binding models, allowing calculation of dissociation constant (Kd ) and binding density (Bmax ) values corresponding to high- and low-affinity DAT binding sites. Scatchard analysis of the saturation binding curves clearly demonstrate the presence of high- and low- affinity binding sites in the NAc, with low-affinity sites comprising 85 to 94% of the binding sites. DAT binding analyses revealed that self-administration of cocaine and a cocaine:heroin combination increased the affinity of the low-affinity site for the cocaine congener RTI-55 compared to saline. These results indicate that the alterations observed following chronic speedball self-administration are likely due to the cocaine component alone; thus further studies are necessary to elaborate upon the synergistic effect of cocaine:heroin combinations on the dopamine system in the NAc.