Circadian Phase-Dependent Protein and Tissular Binding of Three Local Anesthetics (Bupivacaine, Etidocaine, and Mepivacaine) in Mice: A Possible Mechanismof Their Chronotoxicokinetics?

The aim of this study was to investigate the possible influence of the time of administration on bupivacaine (B), etidocaine (E), and mepivacaine (M) protein and tissue (brain and heart) binding. For each anesthetic agent, a single dose of B (20 mg/kg), E (40 mg/kg), or M (60 mg/kg) was administered intraperito-neally at 10:00,16:00,22:00, and 04:00 h. Blood and tissue samples were collected 15 min after drug administration. This study documents significant circadian variations in protein and tissue binding of the three local anesthetic agents. We did not demonstrate a temporal relationship between the respective free and tissue levels. Thus, the temporal variations of free plasma, brain, and heart levels do not seem to be involved in the temporal changes of induced mortality.     

Interplay of Protein Binding Interactions,DNA Mechanics,and Entropy in?DNA Looping Kinetics

DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions.     

Numerous Classes of General Anesthetics Inhibit Etomidate Binding to ��-Aminobutyric Acid Type A (GABAA) Receptors

Enhancement of γ-aminobutyric acid type A receptor (GABA_AR)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA_AR function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA_AR protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA_AR transmembrane domain at the β-α subunit interface; the etomidate analog [³H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, α1Met-236 in the M1 helix and βMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605). Here, we use [³H]azietomidate photolabeling of bovine brain GABA_ARs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of α1Met-236 and βMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA_AR: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.     

Characterization of a High Affinity Folate Binding Protein in Porcine Serum: Ionic Charge, Concentration—Dependent Polymerization and Ligand Binding Mechanism

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-affinity type with upward convex Scatchard plots and Hill coefficients > 1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/l. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing hetecrodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.     

Tissular localization of coumarins in the green alga Dasycladus vermicularis (Scopoli) Krasser: a photoprotective role?

Cell distribution of coumarins, a group of UV-absorbing substances, was analysed by epifluorescence optical microscopy in the green macroalga Dasycladus vermicularis. Maximal concentration of 3,6,7-trihydroxycoumarin (THC), which corresponds to almost 100% of the total coumarins in D. vermicularis, was found in the apical part of the thallus, which is more exposed to solar radiation. At a cell level, two blue, highly fluorescent layers, corresponding to a large accumulation of THC, were found in the internal part of the cell wall and around the vacuolar membrane. The percentage of UV radiation absorbed by each THC layer could be measured from the in vitro total thallus concentration of THC and histological measurements of the layers. The THC layer close to the cell wall absorbed 88% of the incident irradiance at 346 nm (corresponding to the maximum of absorbance of THC in the UVA region), while that close to the vacuole membrane absorbed 87.5%. These results agree with the hypothesis of a natural sunscreen role, significantly reducing harmful UV radiation reaching the cell. Owing to the release of this substance into the medium under different stress conditions, its capacity as a UV filter for other macroalgae has been tested. The ecological relevance of the release process of this UV-absorbing substance in specific environments is discussed.     

Mutations at Beta N265 in γ-Aminobutyric Acid Type A Receptors Alter Both Binding Affinity and Efficacy of Potent Anesthetics

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α− inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15′) on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the βN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between βN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1β2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the β-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1β2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive βN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1β2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in β+/α− interfaces, we found that βN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. βN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics β+/α− anesthetic site occupancy. Our results show that βN265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in β+/α− sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for βN265 in anesthetic binding within the β+/α− transmembrane sites.     

RETRACTION: C-reactive Protein and Metabolic Syndrome in Youth: A Strong Relationship?

Retraction: Note from the Editor‐in‐Chief: This paper is retracted Objective: Metabolic syndrome (MS) is on the rise in youth. As high‐sensitivity C‐reactive protein (hs‐CRP) is associated with cardiovascular/metabolic disorders, we evaluated the association between MS and its components and hs‐CRP in a sample of Brazilian overweight and obese youth. Methods and Procedures: A total of 407 students (229 girls, 273 with excessive weight, 11.3 ± 3.2 years) were evaluated. Measurement included BMI, waist circumference (WC), blood pressure, lipids, insulin, and hs‐CRP. Excessive weight was defined using BMI z ‐score; MS by the modified National Cholesterol Education Program—Adult Treatment Panel III. Results: Subjects were classified into two groups: with MS (n = 72) and without (n = 335). hs‐CRP means and medians were higher in MS group (1.41 mg/l vs. 1.06 mg/l, P < 0.001; 2.21 mg/l vs. 1.23 mg/l, P < 0.001). Associations between hs‐CRP quartiles and insulin resistance (IR) (P < 0.001), MS (P < 0.001), WC (P < 0.000), BMI z‐score (P < 0.001), hypertension (P < 0.001), hypertriglyceridemia (P < 0.001), and low HDL‐c (P = 0.023) were significant; adjustment of hs‐CRP for BMI z‐score eliminated the previous association, except for the number of MS components (nMSc) (P < 0.001). Adjusting for homeostasis model assessment method of IR (HOMA‐IR) did not eliminate the relation between hs‐CRP and MS components. Furthermore, increases in BMI z ‐score and nMSc were associated with an increased hs‐CRP. Excessive weight (odds ratio (OR), 7.9; confidence interval (CI), 4.7–13.4; P = 0.000), hypertension (OR, 2.3; CI, 1.3–4.2; P = 0.003), and hypertriglyceridemia (OR, 2.3; CI, 1.5–3.7; P < 0.001) were independently associated with hs‐CRP. Discussion: In youth, hs‐CRP is strongly related with MS and its components, and is also determined by the body composition. This association indicates a precocious proinflammatory state.     

Reconstitution of the Peridinin–chlorophyll a Protein (PCP): Evidence for Functional Flexibility in Chlorophyll Binding

The coding regions for the N-domain, and full length peridinin–chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was ~86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.     

NSE-Controlled Carboxyl-Terminus of APP Gene Over-Expressing in Transgenic Mice Induces Altered Expressions in Behavior, Aβ-42, and GSK3β Binding Proteins

Summary The amyloid protein precursor (APP) is cleaved in its intramembranous domain by γ-secrease to generate amyloid β and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer’s disease (AD), but it is incompletely understand how APP-C105 overexpression interacts and regulates the brain function and Aβ-42 levels, and whether or not it is associated with the expressions of GSK3β-binding proteins. To test this, transgenic mice expressing NSE-controlled APP-C105 were produced and tested for their above phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in transgenic brains compared with those of non-transgenic brains. In parallel, APP-C105 overexpression resulted in the modulation of the Aβ-42 level, γ-secretase activity, GSK3β-binding proteins including PS1, tau, and β-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Thus, altered expressions of these neuropathological phenotypes in APP-C105 transgenic mice could be useful targets in developing new therapeutic treatments.     

Structural Alerts,Reactive Metabolites,and Protein Covalent Binding: How Reliable Are These Attributes as Predictors of Drug Toxicity?

In an increasing number of cases, a deeper understanding of the biochemical basis for idiosyncratic adverse drug reactions (IADRs) has aided to replace a vague perception of a chemical class effect with a sharper picture of individual molecular peculiarity. Considering that IADRs are too complex to duplicate in a test tube, and their idiosyncratic nature precludes prospective clinical studies, it is currently impossible to predict which new drugs will be associated with a significant incidence of toxicity. Because it is now widely appreciated that reactive metabolites, as opposed to the parent molecules from which they are derived, are responsible for the pathogenesis of some IADRs, the propensity of drug candidates to form reactive metabolites is generally considered a liability. Procedures have been implemented to monitor reactive‐metabolite formation in discovery with the ultimate goal of eliminating or minimizing the liability via rational structural modification of the problematic chemical series. While such mechanistic studies have provided retrospective insight into the metabolic pathways which lead to reactive metabolite formation with toxic compounds, their ability to accurately predict the IADR potential of new drug candidates has been challenged. There are several instances of drugs that form reactive metabolites, but only a fraction thereof cause toxicity. This review article will outline current approaches to evaluate bioactivation potential of new compounds with particular emphasis on the advantages and limitation of these assays. Plausible reason(s) for the excellent safety record of certain drugs susceptible to bioactivation will also be explored and should provide valuable guidance in the use of reactive‐metabolite assessments when nominating drug candidates for development.     

Testate Amoebae (Arcellinida and Euglyphida) vs. Ericoid Mycorrhizal and DSE Fungi: A Possible Novel Interaction in the Mycorrhizosphere of Ericaceous Plants?

Common occurrence of testate amoebae (TA) in the rhizosphere of mycorrhizal plants indicates existence of yet undocumented ecological interactions, involving three distinct groups of organisms: soil protists, mycorrhizal fungi, and their host plants. This tripartite relationship was to date investigated only to a limited extent, despite its probable importance for processes taking place in the mycorrhizosphere. In this study, we (1) explored spectra of different TA genera naturally associated with the rhizoplane of three autochthonous European Rhododendron species, (2) screened natural fungal colonization of the TA shells occupying the rhizoplane of selected rhododendrons, and (3) carried out two in vitro experiments addressing the question whether TA shells may serve as a nutrient source for ericoid mycorrhizal fungi (ErMF) and dark septate endophytes (DSE). Our field observations indicated that TA regularly associated with the rhizoplane of all screened rhododendrons and that ErMF and/or DSE associated with their roots possibly exploited the TA shells as a nutrient source. We were unable to detect any major differences among the TA spectra from the rhizoplanes with respect to the three Rhododendron species. The spectra were dominated by Diplochlamys, Centropyxis, Cyclopyxis, Euglypha, Trinema, and Assulina. Positive, neutral, and negative associations were found for various TA genera × Rhododendron species combinations. The highest fungal colonization was observed in Centropyxidae and Trigonopyxidae, reaching up to 45% of the shells in the case of Trigonopyxis. In the in vitro experiments, both ErMF Rhizoscyphus ericae and DSE Phialocephala fortinii regularly colonized TA shells, utilizing them as a source of nutrients. We hypothesize a complex relationship between ErMF–DSE and TA. If corroborated, it would represent an interesting nutrient loop in the mycorrhizosphere of ericaceous plants.     

Interaction of Double-stranded RNA-dependent Protein Kinase (PKR) with the Death Receptor Signaling Pathway in Amyloid �� (A��)-treated Cells and in APPSLPS1 Knock-in Mice

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APP_SLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP_SLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT⁴⁵¹-PKR and pS¹⁹⁴-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.     

Targeting Fatty Acid Binding Protein (FABP) Anandamide Transporters – A Novel Strategy for Development of Anti-Inflammatory and Anti-Nociceptive Drugs

Fatty acid binding proteins (FABPs), in particular FABP5 and FABP7, have recently been identified by us as intracellular transporters for the endocannabinoid anandamide (AEA). Furthermore, animal studies by others have shown that elevated levels of endocannabinoids resulted in beneficial pharmacological effects on stress, pain and inflammation and also ameliorate the effects of drug withdrawal. Based on these observations, we hypothesized that FABP5 and FABP7 would provide excellent pharmacological targets. Thus, we performed a virtual screening of over one million compounds using DOCK and employed a novel footprint similarity scoring function to identify lead compounds with binding profiles similar to oleic acid, a natural FABP substrate. Forty-eight compounds were purchased based on their footprint similarity scores (FPS) and assayed for biological activity against purified human FABP5 employing a fluorescent displacement-binding assay. Four compounds were found to exhibit approximately 50% inhibition or greater at 10 µM, as good as or better inhibitors of FABP5 than BMS309403, a commercially available inhibitor. The most potent inhibitor, γ-truxillic acid 1-naphthyl ester (ChemDiv 8009-2334), was determined to have K_i value of 1.19±0.01 µM. Accordingly a novel α-truxillic acid 1-naphthyl mono-ester (SB-FI-26) was synthesized and assayed for its inhibitory activity against FABP5, wherein SB-FI-26 exhibited strong binding (K_i 0.93±0.08 µM). Additionally, we found SB-FI-26 to act as a potent anti-nociceptive agent with mild anti-inflammatory activity in mice, which strongly supports our hypothesis that the inhibition of FABPs and subsequent elevation of anandamide is a promising new approach to drug discovery. Truxillic acids and their derivatives were also shown by others to have anti-inflammatory and anti-nociceptive effects in mice and to be the active component of Chinese a herbal medicine (Incarvillea sinensis) used to treat rheumatism and pain in humans. Our results provide a likely mechanism by which these compounds exert their effects.     

The Lengthening of a Giant Protein: When, How, and Why?

Subcommissural organ (SCO)-spondin is a giant glycoprotein of more than 5000 amino acids found in Vertebrata, expressed in the central nervous system and constitutive of Reissner’s fiber. For the first time, in situ hybridization performed on zebrafish (Danio rerio) embryos shows that the gene encoding this protein is expressed transitionally in the floor plate, the ventral midline of the neural tube, and later in the diencephalic third ventricle roof, the SCO. The modular organization of the protein in Echinodermata (Strongylocentrotus purpuratus), Urochordata (Ciona savignyi and C. intestinalis), and Vertebrata (Teleostei, Amphibia, Aves and Mammalia) is also described. As the thrombospondin type 1 repeat motifs represent an increasingly large part of the protein during Deuterostomia evolution, the duplication mechanisms leading to this complex organization are examined. The functional significance of the particularly well-preserved arrangement of the series of SCO-spondin repeat motifs and thombospondin type 1 repeats is discussed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cleavage of Poly(A)-Binding Protein by Coxsackievirus 2A Protease In Vitro and In Vivo: Another Mechanism for Host Protein Synthesis Shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at ⁴⁸²VANTSTQTM↓GPRPAAAAAA⁵⁰⁰, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.