Methamphetamine-induced sensitization differentially alters pCREB and DeltaFosB throughout the limbic circuit of the mammalian brain.

PubMed

McDaid, John; Graham, Martin P; Napier, T Celeste

2006-12-01

Enhancements in behavior that accompany repeated, intermittent administration of abused drugs (sensitization) endure long after drug administration has ceased. Such persistence reflects changes in intracellular signaling cascades and associated gene transcription factors in brain regions that are engaged by abused drugs. This process is not characterized for the most potent psychomotor stimulant, methamphetamine. Using motor behavior as an index of brain state in rats, we verified that five once-daily injections of 2.5 mg/kg methamphetamine induced behavioral sensitization that was demonstrated (expressed) 3 and 14 days later. Using immunoblot procedures, limbic brain regions implicated in behavioral sensitization were assayed for extracellular signal-regulated kinase and its phosphorylated form (pERK/ERK, a signal transduction kinase), cAMP response element binding protein and its phosphorylated form (pCREB/CREB, a constitutively expressed transcriptional regulator), and DeltaFosB (a long-lasting transcription factor). pERK, ERK, and CREB levels were not changed for any region assayed. In the ventral tegmental area, pCREB and DeltaFosB also were not changed. pCREB (activated CREB) was elevated in the frontal cortex at 3 days withdrawal, but not at 14 days. pCREB levels were decreased at 14 days withdrawal in the nucleus accumbens and ventral pallidum. Accumbal and pallidal levels of DeltaFosB were increased at 3 days withdrawal, and this increase persisted to 14 days in the pallidum. Thus, only the ventral pallidum showed changes in molecular processes that consistently correlated with motor sensitization, revealing that this region may be associated with this enduring behavioral phenotype initiated by methamphetamine. The present findings expand our understanding of the neuroanatomical and molecular substrates that may play a role in the persistence of druginduced sensitization.

Stress-enhanced gelation: a dynamic nonlinearity of elasticity.

PubMed

Yao, Norman Y; Broedersz, Chase P; Depken, Martin; Becker, Daniel J; Pollak, Martin R; Mackintosh, Frederick C; Weitz, David A

2013-01-04

A hallmark of biopolymer networks is their sensitivity to stress, reflected by pronounced nonlinear elastic stiffening. Here, we demonstrate a distinct dynamical nonlinearity in biopolymer networks consisting of filamentous actin cross-linked by α-actinin-4. Applied stress delays the onset of relaxation and flow, markedly enhancing gelation and extending the regime of solidlike behavior to much lower frequencies. We show that this macroscopic network response can be accounted for at the single molecule level by the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior.

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity

DOE PAGES

Luca, Vincent C.; Kim, Byoung Choul; Ge, Chenghao; ...

2017-03-02

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes uponmore » Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. In conclusion, this mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.« less

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luca, Vincent C.; Kim, Byoung Choul; Ge, Chenghao

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes uponmore » Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. In conclusion, this mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.« less

Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction.

PubMed

Jacobsen, Michael T; Fairhead, Michael; Fogelstrand, Per; Howarth, Mark

2017-08-17

Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

PubMed Central

Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

2015-01-01

Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

PubMed

González, Andrea; Nova, Esteban; Del Campo, Miguel; Manubens, Augusto; De Ioannes, Alfredo; Ferreira, Jorge; Becker, María Inés

2017-12-01

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Orbitofrontal Dopamine Depletion Upregulates Caudate Dopamine and Alters Behavior via Changes in Reinforcement Sensitivity

PubMed Central

Cardinal, R. N.; Rygula, R.; Hong, Y. T.; Fryer, T. D.; Sawiak, S. J.; Ferrari, V.; Cockcroft, G.; Aigbirhio, F. I.; Robbins, T. W.; Roberts, A. C.

2014-01-01

Schizophrenia is associated with upregulation of dopamine (DA) release in the caudate nucleus. The caudate has dense connections with the orbitofrontal cortex (OFC) via the frontostriatal loops, and both areas exhibit pathophysiological change in schizophrenia. Despite evidence that abnormalities in dopaminergic neurotransmission and prefrontal cortex function co-occur in schizophrenia, the influence of OFC DA on caudate DA and reinforcement processing is poorly understood. To test the hypothesis that OFC dopaminergic dysfunction disrupts caudate dopamine function, we selectively depleted dopamine from the OFC of marmoset monkeys and measured striatal extracellular dopamine levels (using microdialysis) and dopamine D2/D3 receptor binding (using positron emission tomography), while modeling reinforcement-related behavior in a discrimination learning paradigm. OFC dopamine depletion caused an increase in tonic dopamine levels in the caudate nucleus and a corresponding reduction in D2/D3 receptor binding. Computational modeling of behavior showed that the lesion increased response exploration, reducing the tendency to persist with a recently chosen response side. This effect is akin to increased response switching previously seen in schizophrenia and was correlated with striatal but not OFC D2/D3 receptor binding. These results demonstrate that OFC dopamine depletion is sufficient to induce striatal hyperdopaminergia and changes in reinforcement learning relevant to schizophrenia. PMID:24872570

DNA Binding Hydroxyl Radical Probes.

PubMed

Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

2012-01-01

The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

Dopamine D3 Receptor Availability Is Associated with Inflexible Decision Making.

PubMed

Groman, Stephanie M; Smith, Nathaniel J; Petrullli, J Ryan; Massi, Bart; Chen, Lihui; Ropchan, Jim; Huang, Yiyun; Lee, Daeyeol; Morris, Evan D; Taylor, Jane R

2016-06-22

Dopamine D2/3 receptor signaling is critical for flexible adaptive behavior; however, it is unclear whether D2, D3, or both receptor subtypes modulate precise signals of feedback and reward history that underlie optimal decision making. Here, PET with the radioligand [(11)C]-(+)-PHNO was used to quantify individual differences in putative D3 receptor availability in rodents trained on a novel three-choice spatial acquisition and reversal-learning task with probabilistic reinforcement. Binding of [(11)C]-(+)-PHNO in the midbrain was negatively related to the ability of rats to adapt to changes in rewarded locations, but not to the initial learning. Computational modeling of choice behavior in the reversal phase indicated that [(11)C]-(+)-PHNO binding in the midbrain was related to the learning rate and sensitivity to positive, but not negative, feedback. Administration of a D3-preferring agonist likewise impaired reversal performance by reducing the learning rate and sensitivity to positive feedback. These results demonstrate a previously unrecognized role for D3 receptors in select aspects of reinforcement learning and suggest that individual variation in midbrain D3 receptors influences flexible behavior. Our combined neuroimaging, behavioral, pharmacological, and computational approach implicates the dopamine D3 receptor in decision-making processes that are altered in psychiatric disorders. Flexible decision-making behavior is dependent upon dopamine D2/3 signaling in corticostriatal brain regions. However, the role of D3 receptors in adaptive, goal-directed behavior has not been thoroughly investigated. By combining PET imaging with the D3-preferring radioligand [(11)C]-(+)-PHNO, pharmacology, a novel three-choice probabilistic discrimination and reversal task and computational modeling of behavior in rats, we report that naturally occurring variation in [(11)C]-(+)-PHNO receptor availability relates to specific aspects of flexible decision making. We confirm these relationships using a D3-preferring agonist, thus identifying a unique role of midbrain D3 receptors in decision-making processes. Copyright © 2016 the authors 0270-6474/16/366732-10$15.00/0.

NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.

PubMed

Singh-Taylor, A; Molet, J; Jiang, S; Korosi, A; Bolton, J L; Noam, Y; Simeone, K; Cope, J; Chen, Y; Mortazavi, A; Baram, T Z

2018-03-01

Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

The Small GTP-Binding Protein Rhes Influences Nigrostriatal-Dependent Motor Behavior During Aging.

PubMed

Pinna, Annalisa; Napolitano, Francesco; Pelosi, Barbara; Di Maio, Anna; Wardas, Jadwiga; Casu, Maria Antonietta; Costa, Giulia; Migliarini, Sara; Calabresi, Paolo; Pasqualetti, Massimo; Morelli, Micaela; Usiello, Alessandro

2016-04-01

Here we aimed to evaluate: (1) Rhes mRNA expression in mouse midbrain, (2) the effect of Rhes deletion on the number of dopamine neurons, (3) nigrostriatal-sensitive behavior during aging in knockout mice. Radioactive in situ hybridization was assessed in adult mice. The beam-walking test was executed in 3-, 6- and 12-month-old mice. Immunohistochemistry of midbrain tyrosine hydroxylase (TH)-positive neurons was performed in 6- and 12-month-old mice. Rhes mRNA is expressed in TH-positive neurons of SNpc and the ventral tegmental area. Moreover, lack of Rhes leads to roughly a 20% loss of nigral TH-positive neurons in both 6- and 12-month-old mutants, when compared with their age-matched controls. Finally, lack of Rhes triggers subtle alterations in motor performance and coordination during aging. Our findings indicate a fine-tuning role of Rhes in regulating the number of TH-positive neurons of the substantia nigra and nigrostriatal-sensitive motor behavior during aging. © 2016 International Parkinson and Movement Disorder Society.

Neurosteroid Influences on Sensitivity to Ethanol

PubMed Central

Helms, Christa M.; Rossi, David J.; Grant, Kathleen A.

2011-01-01

This review will highlight a variety of mechanisms by which neurosteroids affect sensitivity to ethanol, including physiological states associated with activity of the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes, and the effects of chronic exposure to ethanol, in addition to behavioral implications. To date, γ-aminobutyric acid (GABAA) receptor mechanisms are a major focus of the modulation of ethanol effects by neuroactive steroids. While NMDA receptor mechanisms are gaining prominence in the literature, these complex data would be best discussed separately. Accordingly, GABAA receptor mechanisms are emphasized in this review with brief mention of some NMDA receptor mechanisms to point out contrasting neuroactive steroid pharmacology. Overall, the data suggest that neurosteroids are virtually ubiquitous modulators of inhibitory neurotransmission. Neurosteroids appear to affect sensitivity to ethanol in specific brain regions and, consequently, specific behavioral tests, possibly related to the efficacy and potency of ethanol to potentiate the release of GABA and increase neurosteroid concentrations. Although direct interaction of ethanol and neuroactive steroids at common receptor binding sites has been suggested in some studies, this proposition is still controversial. It is currently difficult to assign a specific mechanism by which neuroactive steroids could modulate the effects of ethanol in particular behavioral tasks. PMID:22654852

A first-principles study on adsorption behaviors of pristine and Li-decorated graphene sheets toward hydrazine molecules

NASA Astrophysics Data System (ADS)

Zeng, Huadong; Cheng, Xinlu; Wang, Wei

2018-03-01

The adsorption behaviors and properties of hydrazine (N2H4) molecules on pristine and Li-decorated graphene sheets were investigated by means of first-principles based on density functional theory. We systematically analyzed the optimal geometry, average binding energy, charge transfer, charge density difference and density of states of N2H4 molecules adsorbed on pristine and Li-decorated graphene sheets. It is found that the interaction between single N2H4 molecule and pristine graphene is weak physisorption with the low binding energy of -0.026 eV, suggesting that the pristine graphene sheet is insensitive to the presence of N2H4 molecule. However, it is markedly enhanced after lithium decoration with the high binding energy of -1.004 eV, verifying that the Li-decorated graphene sheet is significantly sensitive to detect N2H4 molecule. Meanwhile, the effects of the concentrations of N2H4 molecules on two different substrates were studied detailedly. For pristine graphene substrate, the average binding energy augments apparently with increasing the number of N2H4 molecules, which is mainly attributed to the van der Waals interactions and hydrogen bonds among N2H4 clusters. Li-decorated graphene sheet has still a strong affinity to N2H4 molecules despite the corresponding average binding energy emerges a contrary tendency. Overall, Li-decorated graphene sheet could be considered as a potential gas sensor in field of hydrazine molecules.

Understanding the length dependence of molecular junction thermopower.

PubMed

Karlström, Olov; Strange, Mikkel; Solomon, Gemma C

2014-01-28

Thermopower of molecular junctions is sensitive to details in the junction and may increase, decrease, or saturate with increasing chain length, depending on the system. Using McConnell's theory for exponentially suppressed transport together with a simple and easily interpretable tight binding model, we show how these different behaviors depend on the molecular backbone and its binding to the contacts. We distinguish between resonances from binding groups or undercoordinated electrode atoms, and those from the periodic backbone. It is demonstrated that while the former gives a length-independent contribution to the thermopower, possibly changing its sign, the latter determines its length dependence. This means that the question of which orbitals from the periodic chain that dominate the transport should not be inferred from the sign of the thermopower but from its length dependence. We find that the same molecular backbone can, in principle, show four qualitatively different thermopower trends depending on the binding group: It can be positive or negative for short chains, and it can either increase or decrease with length.

Enhancement of Behavioral Sensitization, Anxiety-Like Behavior, and Hippocampal and Frontal Cortical CREB Levels Following Cocaine Abstinence in Mice Exposed to Cocaine during Adolescence

PubMed Central

Valzachi, Maria Cristina; Teodorov, Elizabeth; Marcourakis, Tania; Bailey, Alexis; Camarini, Rosana

2013-01-01

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system. PMID:24205196

Sex differences in neuromuscular androgen receptor expression and sociosexual behavior in a sex changing fish

PubMed Central

Pradhan, Devaleena S.; Thonkulpitak, Kevin; Drilling, Cathleen; Black, Michael; Grober, Matthew S.

2017-01-01

Androgen signaling, via receptor binding, is critical for regulating the physiological and morphological foundations of male-typical reproductive behavior in vertebrates. Muscles essential for male courtship behavior and copulation are highly sensitive to androgens. Differences in the distribution and density of the androgen receptor (AR) are important for maintaining dimorphic musculature and thus may provide for anatomical identification of sexually selected traits. In Lythrypnus dalli, a bi-directional hermaphroditic teleost fish, both sexes produce agonistic approach displays, but reproductive behavior is sexually dimorphic. The male-specific courtship behavior is characterized by rapid jerky movements (involving dorsal fin erection) towards a female or around their nest. Activation of the supracarinalis muscle is involved in dorsal fin contributions to both agonistic and sociosexual behavior in other fishes, suggesting that differences in goby sexual behavior may be reflected in sexual dimorphism in AR signaling in this muscle. We examined sex differences in the local distribution of AR in supracarinalis muscle and spinal cord. Our results demonstrate that males do express more AR in the supracarinalis muscle relative to females, but there was no sex difference in the number of spinal motoneurons expressing AR. Interestingly, AR expression in the supracarinalis muscle was also related to rates of sociosexual behavior in males, providing evidence that sexual selection may influence muscle androgenic sensitivity to enhance display vigor. Sex differences in the distribution and number of cells expressing AR in the supracarinalis muscle may underlie the expression of dimorphic behaviors in L. dalli. PMID:28520775

NMR studies of DNA oligomers and their interactions with minor groove binding ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fagan, Patricia A.

1996-05-01

The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1more » ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.« less

Electronic transport in smectic liquid crystals

NASA Astrophysics Data System (ADS)

Shiyanovskaya, I.; Singer, K. D.; Twieg, R. J.; Sukhomlinova, L.; Gettwert, V.

2002-04-01

Time-of-flight measurements of transient photoconductivity have revealed bipolar electronic transport in phenylnaphthalene and biphenyl liquid crystals (LC), which exhibit several smectic mesophases. In the phenylnaphthalene LC, the hole mobility is significantly higher than the electron mobility and exhibits different temperature and phase behavior. Electron mobility in the range ~10-5 cm2/V s is temperature activated and remains continuous at the phase transitions. However, hole mobility is nearly temperature independent within the smectic phases, but is very sensitive to smectic order, 10-3 cm2/V s in the smectic-B (Sm-B) and 10-4 cm2/V s in the smectic-A (Sm-A) mesophases. The different behavior for holes and electron transport is due to differing transport mechanisms. The electron mobility is apparently controlled by rate-limiting multiple shallow trapping by impurities, but hole mobility is not. To explain the lack of temperature dependence for hole mobility within the smectic phases we consider two possible polaron transport mechanisms. The first mechanism is based on the hopping of Holstein small polarons in the nonadiabatic limit. The polaron binding energy and transfer integral values, obtained from the model fit, turned out to be sensitive to the molecular order in smectic mesophases. A second possible scenario for temperature-independent hole mobility involves the competion between two different polaron mechanisms involving so-called nearly small molecular polarons and small lattice polarons. Although the extracted transfer integrals and binding energies are reasonable and consistent with the model assumptions, the limited temperature range of the various phases makes it difficult to distinguish between any of the models. In the biphenyl LCs both electron and hole mobilities exhibit temperature activated behavior in the range of 10-5 cm2/V s without sensitivity to the molecular order. The dominating transport mechanism is considered as multiple trapping in the impurity sites. Temperature-activated mobility was treated within the disorder formalism, and activation energy and width of density of states have been calculated.

Optical Control of Dopamine Receptors Using a Photoswitchable Tethered Inverse Agonist.

PubMed

Donthamsetti, Prashant C; Winter, Nils; Schönberger, Matthias; Levitz, Joshua; Stanley, Cherise; Javitch, Jonathan A; Isacoff, Ehud Y; Trauner, Dirk

2017-12-27

Family A G protein-coupled receptors (GPCRs) control diverse biological processes and are of great clinical relevance. Their archetype rhodopsin becomes naturally light sensitive by binding covalently to the photoswitchable tethered ligand (PTL) retinal. Other GPCRs, however, neither bind covalently to ligands nor are light sensitive. We sought to impart the logic of rhodopsin to light-insensitive Family A GPCRs in order to enable their remote control in a receptor-specific, cell-type-specific, and spatiotemporally precise manner. Dopamine receptors (DARs) are of particular interest for their roles in motor coordination, appetitive, and aversive behavior, as well as neuropsychiatric disorders such as Parkinson's disease, schizophrenia, mood disorders, and addiction. Using an azobenzene derivative of the well-known DAR ligand 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), we were able to rapidly, reversibly, and selectively block dopamine D1 and D2 receptors (D1R and D2R) when the PTL was conjugated to an engineered cysteine near the dopamine binding site. Depending on the site of tethering, the ligand behaved as either a photoswitchable tethered neutral antagonist or inverse agonist. Our results indicate that DARs can be chemically engineered for selective remote control by light and provide a template for precision control of Family A GPCRs.

Effect of melatonin on methamphetamine- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity and methamphetamine-induced behavioral sensitization.

PubMed

Itzhak, Y; Martin, J L; Black, M D; Ali, S F

1998-06-01

Methamphetamine (METH)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity is thought to be associated with the formation of free radicals. Since evidence suggests that melatonin may act as a free radical scavenger and antioxidant, the present study was undertaken to investigate the effect of melatonin on METH- and MPTP-induced neurotoxicity. In addition, the effect of melatonin on METH-induced locomotor sensitization was investigated. The administration of METH (5 mg kg(-1) x 3) or MPTP (20 mg kg(-1) x 3) to Swiss Webster mice resulted in 45-57% depletion in the content of striatal dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 57-59% depletion in dopamine transporter binding sites. The administration of melatonin (10 mg kg(-1)) before each of the three injections of the neurotoxic agents (on day 1), and thereafter for two additional days, afforded a full protection against METH-induced depletion of dopamine and its metabolites and dopamine transporter binding sites. In addition, melatonin significantly diminished METH-induced hyperthermia. However, the treatment with melatonin had no significant effect on MPTP-induced depletion of the dopaminergic markers tested. In the set of behavioral experiments, we found that the administration of 1 mg kg(-1) METH to Swiss Webster mice for 5 days resulted in marked locomotor sensitization to a subsequent challenge injection of METH, as well as context-dependent sensitization (conditioning). The pretreatment with melatonin (10 mg kg(-1)) prevented neither the sensitized response to METH nor the development of conditioned locomotion. Results of the present study indicate that melatonin has a differential effect on the dopaminergic neurotoxicity produced by METH and MPTP. Since it is postulated that METH-induced hyperthermia is related to its neurotoxic effect, while regulation of body temperature is unrelated to MPTP-induced neurotoxicity or METH-induced locomotor sensitization, the protective effect of melatonin observed in the present study may be due primarily to diminishing METH-induced hyperthermia.

Inferring Diffusion Dynamics from FCS in Heterogeneous Nuclear Environments

PubMed Central

Tsekouras, Konstantinos; Siegel, Amanda P.; Day, Richard N.; Pressé, Steve

2015-01-01

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show—first using synthetic data—that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell’s nucleus as well as 2) in the cell’s cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins. PMID:26153697

The Rewarding and Locomotor-Sensitizing Effects of Repeated Cocaine Administration are Distinct and Separable in Mice

PubMed Central

Riday, Thorfinn T.; Kosofsky, Barry E.; Malanga, C.J.

2011-01-01

Repeated psychostimulant exposure progressively increases their potency to stimulate motor activity in rodents. This behavioral or locomotor sensitization is considered a model for some aspects of drug addiction in humans, particularly drug craving during abstinence. However, the role of increased motor behavior in drug reward remains incompletely understood. Intracranial self-stimulation (ICSS) was measured concurrently with locomotor activity to determine if acute intermittent cocaine administration had distinguishable effects on motor behavior and perception of brain stimulation-reward (BSR) in the same mice. Sensitization is associated with changes in neuronal activity and glutamatergic neurotransmission in brain reward circuitry. Expression of AMPA receptor subunits (GluR1 and GluR2) and CRE binding protein (CREB) was measured in the ventral tegmental area (VTA), dorsolateral striatum (STR) and nucleus accumbens (NAc) before and after a sensitizing regimen of cocaine, with and without ICSS. Repeated cocaine administration sensitized mice to its locomotor stimulating effects but not its ability to potentiate BSR. ICSS increased GluR1 in the VTA but not NAc or STR, demonstrating selective changes in protein expression with electrical stimulation of discrete brain structures. Repeated cocaine reduced GluR1, GluR2 and CREB expression in the NAc, and reductions of GluR1 and GluR2 but not CREB were further enhanced by ICSS. These data suggest that the effects of repeated cocaine exposure on reward and motor processes are dissociable in mice, and that reduction of excitatory neurotransmission in the NAc may predict altered motor function independently from changes in reward perception. PMID:22197517

Long-term alterations in vulnerability to addiction to drugs of abuse and in brain gene expression after early life ethanol exposure.

PubMed

Barbier, Estelle; Pierrefiche, Olivier; Vaudry, David; Vaudry, Hubert; Daoust, Martine; Naassila, Mickaël

2008-12-01

Exposure to ethanol early in life can have long-lasting implications on brain function and drug of abuse response later in life. The present study investigated in rats, the long-term consequences of pre- and postnatal (early life) ethanol exposure on drug consumption/reward and the molecular targets potentially associated with these behavioral alterations. Since a relationship has been demonstrated between heightened drugs intake and susceptibility to drugs-induced locomotor activity/sensitization, anxiolysis, we tested these behavioral responses, depending on the drug, in control and early life ethanol-exposed animals. Our results show that progeny exposed to early life ethanol displayed increased consumption of ethanol solutions and increased sensitivity to cocaine rewarding effects assessed in the conditioned place preference test. Offspring exposed to ethanol were more sensitive to the anxiolytic effect of ethanol and the increased sensitivity could, at least in part, explain the alteration in the consumption of ethanol for its anxiolytic effects. In addition, the sensitivity to hypothermic effects of ethanol and ethanol metabolism were not altered by early life ethanol exposure. The sensitization to cocaine (20 mg/kg) and to amphetamine (1.2 mg/kg) was increased after early life ethanol exposure and, could partly explain, an increase in the rewarding properties of psychostimulants. Gene expression analysis revealed that expression of a large number of genes was altered in brain regions involved in the reinforcing effects of drugs of abuse. Dopaminergic receptors and transporter binding sites were also down-regulated in the striatum of ethanol-exposed offspring. Such long-term neurochemical alterations in transmitter systems and in the behavioral responses to ethanol and other drugs of abuse may confer an increased liability for addiction in exposed offspring.

Fluorimetric assay of interaction of protein with ferrofluids

NASA Astrophysics Data System (ADS)

Mallik, Dhriti; Mir, Aparna; Bhattacharya, Soumya; Nayar, Suprabha

2011-01-01

Magnetic iron oxide nanoparticles are inherently biocompatible and are amenable to post synthesis surface modification, making them excellent candidates for many important applications. If the above can be achieved in a single-step i.e., in situ synthesis and functionalization, the results are expected to be more dramatic for sensitive detection of biomolecules. For any application, it is necessary to confer a high level of binding specificity through surface chemistry, which can be introduced by using biological moieties that possess lock-and-key interactions, like those observed in antibody-antigen and enzyme-substrate recognition. In this paper, we have synthesized water based ferrofluids with serum albumin, the major protein component of blood. A series of other ferrofluids using different biocompatible polymers have also been studied with respect to their size determined by transmission electron microscopy, magnetic behavior with the aid of vibrating sample magnetometry and binding capability to bovine serum albumin by quenching of its native fluorescence. From our results, it can be inferred that binding has taken place between magnetic particles and biomolecules, the binding constants of which indirectly reveal the efficiency of the interaction.

Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity

PubMed Central

Eisenstein, Sarah A.; Gredysa, Danuta M.; Antenor–Dorsey, Jo Ann; Green, Leonard; Arbeláez, Ana Maria; Koller, Jonathan M.; Black, Kevin J.; Perlmutter, Joel S.; Moerlein, Stephen M.; Hershey, Tamara

2015-01-01

Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30) and non-obese (n = 20; body mass index<30) adults were assessed for percent body fat with dual-energy X-ray absorptiometry and for β-cell function using disposition index. Choice of larger, but delayed or less certain, monetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methyl)benperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting). Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting). In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding and metabolic factors, including β-cell function, interact to affect reward discounting in humans. PMID:26192187

Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity.

PubMed

Eisenstein, Sarah A; Gredysa, Danuta M; Antenor-Dorsey, Jo Ann; Green, Leonard; Arbeláez, Ana Maria; Koller, Jonathan M; Black, Kevin J; Perlmutter, Joel S; Moerlein, Stephen M; Hershey, Tamara

2015-01-01

Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30) and non-obese (n = 20; body mass index<30) adults were assessed for percent body fat with dual-energy X-ray absorptiometry and for β-cell function using disposition index. Choice of larger, but delayed or less certain, monetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methyl)benperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting). Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting). In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding and metabolic factors, including β-cell function, interact to affect reward discounting in humans.

Effects of flow changes on radiotracer binding: Simultaneous measurement of neuroreceptor binding and cerebral blood flow modulation.

PubMed

Sander, Christin Y; Mandeville, Joseph B; Wey, Hsiao-Ying; Catana, Ciprian; Hooker, Jacob M; Rosen, Bruce R

2017-01-01

The potential effects of changes in blood flow on the delivery and washout of radiotracers has been an ongoing question in PET bolus injection studies. This study provides practical insight into this topic by experimentally measuring cerebral blood flow (CBF) and neuroreceptor binding using simultaneous PET/MRI. Hypercapnic challenges (7% CO 2 ) were administered to non-human primates in order to induce controlled increases in CBF, measured with pseudo-continuous arterial spin labeling. Simultaneously, dopamine D 2 /D 3 receptor binding of [ 11 C]raclopride or [ 18 F]fallypride was monitored with dynamic PET. Experiments showed that neither time activity curves nor quantification of binding through binding potentials ( BP ND ) were measurably affected by CBF increases, which were larger than two-fold. Simulations of experimental procedures showed that even large changes in CBF should have little effect on the time activity curves of radiotracers, given a set of realistic assumptions. The proposed method can be applied to experimentally assess the flow sensitivity of other radiotracers. Results demonstrate that CBF changes, which often occur due to behavioral tasks or pharmacological challenges, do not affect PET [ 11 C]raclopride or [ 18 F]fallypride binding studies and their quantification. The results from this study suggest flow effects may have limited impact on many PET neuroreceptor tracers with similar properties.

Three essays on the links between agriculture and energy policies in the U.S

NASA Astrophysics Data System (ADS)

Whistance, Jarrett

The first essay develops and applies a structural, partial equilibrium model of United States biomass supply and demand. The aim is to examine the biomass price and expenditure effects of domestic biofuel policies. The results indicate that the cellulosic biofuel sub-mandate alone could increase biomass prices by an average of 50% to 100% over the baseline values. Biomass expenditures by sectors competing with biofuel producers increase by an average of 26% relative to the baseline suggesting those sectors cannot fully shift away from biomass energy sources. A sensitivity analysis focusing on supply response indicates that the results are not very sensitive to the supply elasticity. This study contributes to the literature by providing policymakers and other energy policy stakeholders with a forward looking analysis of potential policy effects on the U.S. biomass market. The second essay develops a similar type of model applied toward the domestic and international petroleum and petroleum products markets as well as the domestic biofuel market and the domestic light-duty vehicle sector. The goal is to investigate the impact of CAFE standards and alternative-fuel vehicle production incentives on the biofuel market and RFS compliance, in particular. The results suggest that holding CAFE standards at the 2010 level could significantly reduce the blendwall problem in the U.S. ethanol market. Furthermore, the alternative fuel production incentives appear to have only minimal effects. However, there is much uncertainty surrounding the appropriate level of automaker response to those incentives, and a sensitivity analysis indicates the model is fairly sensitive to the assumed level of response. The third essay highlights a few of the theories put forth regarding the expected price behavior of Renewable Identification Numbers (RINs). The theories are tested both observationally and empirically with a dataset containing daily RIN price observations going back to January 2009. The behavior does not always match expectations, although the exact causes remain uncertain. In addition, the information provided by RIN prices is used to test the implications of a binding renewable fuel standard (RFS) versus a non-binding RFS on the ethanol-gasoline price relationship. Cointegration tests provide some evidence that the relationship between conventional ethanol and gasoline prices at the wholesale level is weaker in the presence of a binding RFS.

Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

PubMed Central

Harwood, V J; Gordon, A S

1994-01-01

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes. Images PMID:8031076

Scaling laws for nanoFET sensors

NASA Astrophysics Data System (ADS)

Zhou, Fu-Shan; Wei, Qi-Huo

2008-01-01

The sensitive conductance change of semiconductor nanowires and carbon nanotubes in response to the binding of charged molecules provides a novel sensing modality which is generally denoted as nanoFET sensors. In this paper, we study the scaling laws of nanoplate FET sensors by simplifying nanoplates as random resistor networks with molecular receptors sitting on lattice sites. Nanowire/tube FETs are included as the limiting cases where the device width goes small. Computer simulations show that the field effect strength exerted by the binding molecules has significant impact on the scaling behaviors. When the field effect strength is small, nanoFETs have little size and shape dependence. In contrast, when the field effect strength becomes stronger, there exists a lower detection threshold for charge accumulation FETs and an upper detection threshold for charge depletion FET sensors. At these thresholds, the nanoFET devices undergo a transition between low and large sensitivities. These thresholds may set the detection limits of nanoFET sensors, while they could be eliminated by designing devices with very short source-drain distance and large width.

Model mice for mild-form glycine encephalopathy: behavioral and biochemical characterizations and efficacy of antagonists for the glycine binding site of N-methyl D-aspartate receptor.

PubMed

Kojima-ishii, Kanako; Kure, Shigeo; Ichinohe, Akiko; Shinka, Toshikatsu; Narisawa, Ayumi; Komatsuzaki, Shoko; Kanno, Junnko; Kamada, Fumiaki; Aoki, Yoko; Yokoyama, Hiroyuki; Oda, Masaya; Sugawara, Taku; Mizoi, Kazuo; Nakahara, Daiichiro; Matsubara, Yoichi

2008-09-01

Glycine encephalopathy (GE) is caused by an inherited deficiency of the glycine cleavage system (GCS) and characterized by accumulation of glycine in body fluids and various neurologic symptoms. Coma and convulsions develop in neonates in typical GE while psychomotor retardation and behavioral abnormalities in infancy and childhood are observed in mild GE. Recently, we have established a transgenic mouse line (low-GCS) with reduced GCS activity (29% of wild-type (WT) C57BL/6) and accumulation of glycine in the brain (Stroke, 2007; 38:2157). The purpose of the present study is to characterize behavioral features of the low-GCS mouse as a model of mild GE. Two other transgenic mouse lines were also analyzed: high-GCS mice with elevated GCS activity and low-GCS-2 mice with reduced GCS activity. As compared with controls, low-GCS mice manifested increased seizure susceptibility, aggressiveness and anxiety-like activity, which resembled abnormal behaviors reported in mild GE, whereas high-GCS mice were less sensitive to seizures, hypoactive and less anxious. Antagonists for the glycine-binding site of the N-methyl-D-aspartate receptor significantly ameliorated elevated locomotor activity and seizure susceptibility in the low-GCS mice. Our results suggest the usefulness of low-GCS mice as a mouse model for mild GE and a novel therapeutic strategy.

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse.

PubMed

Jacobsen, Jacob P R; Plenge, Per; Sachs, Benjamin D; Pehrson, Alan L; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L; Dalvi, Prachiti; Robinson, Taylor J; O'Neill, Sharon P; Khoo, King S; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G

2014-12-01

Escitalopram appears to be a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, there by curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and hence anti-depressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram's inhibition here of. Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Recombinant generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). We generated mice expressing either the wild-type human SERT (hSERT(WT)) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT(ALI/VFL+SI/TT)). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HTExt levels. Escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small and tended to be enhanced by R-citalopram co-administration. We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram.

On the intra- and interband plasmon modes in doped armchair graphene nanoribbons

NASA Astrophysics Data System (ADS)

Hoi, Bui Dinh; Davoudiniya, Masoumeh; Yarmohammadi, Mohsen

2018-01-01

With the help of the simple tight-binding Hamiltonian and Green's function technique, we study how intraband and interband plasmon modes of both semiconducting and metallic armchair graphene nanoribbons are influenced by the width, chemical doping, and incident momentum direction. In particular, we investigate the behavior of the frequency-dependent susceptibility when the system is exposed to photons or electrons. Injecting electrons by doping creates a new collective mode due to new states between the valence and conduction bands corresponding to intraband transition for which the effect of ribbon width on these transitions in the semiconducting case is much more sensitive than metallic ones. Furthermore, some critical chemical potential and momentum values for both intraband and interband modes lead to different behaviors for resonant peaks. Another remarkable point is the high sensitivity of intraband plasmons to the direction of incident momentum. In particular, the susceptibility of doped nanoribbons vanishes at perpendicular directions, i.e., the intraband plasmons disappear.

Effects of buspirone, diazepam, and zolpidem on open field behavior, and brain [3H]muscimol binding after buspirone pretreatment.

PubMed

Siemiatkowski, M; Sienkiewicz-Jarosz, H; Członkowska, A I; Bidziński, A; Płaźnik, A

2000-07-01

The effects of 5-HT(1A) receptor agonist buspirone, a nonselective (diazepam), and a selective (zolpidem) GABA(A) receptor agonist were compared in the open field test of neophobia. Unhabituated rats were pretreated with the drugs once, prior to a first exposure to the open field, and their behavior was recorded both during this test and during a second trial 24 h later. It has been hypothesized that the decrease in exploratory activity observed during the second test session may be considered an adaptive reaction to the first day aversive experience (neophobia). If so, a selective modulation of 5-HT and GABA systems activity during the test could bring about significant changes in animal behavior on the retest. Buspirone at the lowest dose of 0.3 mg/kg revealed anxiolytic-like properties on the first day, whereas the action of diazepam and zolpidem was modulated by the dose-related sedative effect. At the dose of 2.4 mg/kg buspirone elicited delayed in time anxiolytic-like action, i.e., produced the antithigmotactic effect during the retrial 24 h later. Diazepam and zolpidem failed to exhibit similar profile of action. Autoradiography of [3H]muscimol binding after pretreatment of rats with buspirone showed a significant increase in the selective radioligand binding within the frontal cortex and a similar, near-significant tendency in the dentate gyrus of the hippocampus. The behavioral data validate buspirone as important drug for the treatment of anxiety disorders, devoid of disruptive influence on motor and cognitive processes. The open field test, as modified by us, appeared sensitive in distinguishing the behavioral profiles of action of different anxiolytic compounds, including 5-HT(1A) receptor agonist. The present results support the assumption that reduced turnover of 5-HT due to stimulation of 5-HT(1A) autoreceptors, may bring about changes in GABA(A) receptor system activity, in some brain structures, leading to the anxiolytic effect.

Graphene-Based Liquid-Gated Field Effect Transistor for Biosensing: Theory and Experiments

PubMed Central

Reiner-Rozman, Ciril; Larisika, Melanie; Nowak, Christoph; Knoll, Wolfgang

2015-01-01

We present an experimental and theoretical characterization for reduced Graphene-Oxide (rGO) based FETs used for biosensing applications. The presented approach shows a complete result analysis and theoretically predictable electrical properties. The formulation was tested for the analysis of the device performance in the liquid gate mode of operation with variation of the ionic strength and pH-values of the electrolytes in contact with the FET. The dependence on the Debye length was confirmed experimentally and theoretically, utilizing the Debye length as a working parameter and thus defining the limits of applicability for the presented rGO-FETs. Furthermore, the FETs were tested for the sensing of biomolecules (bovine serum albumin (BSA) as reference) binding to gate-immobilized anti-BSA antibodies and analyzed using the Langmuir binding theory for the description of the equilibrium surface coverage as a function of the bulk (analyte) concentration. The obtained binding coefficients for BSA are found to be same as in results from literature, hence confirming the applicability of the devices. The FETs used in the experiments were fabricated using wet-chemically synthesized graphene, displaying high electron and hole mobility (μ) and provide the strong sensitivity also for low potential changes (by change of pH, ion concentration, or molecule adsorption). The binding coefficient for BSA-anti-BSA interaction shows a behavior corresponding to the Langmuir adsorption theory with a Limit of Detection (LOD) in the picomolar concentration range. The presented approach shows high reproducibility and sensitivity and a good agreement of the experimental results with the calculated data. PMID:25791463

Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations

PubMed Central

Koulgi, Shruti; Achalere, Archana; Sonavane, Uddhavesh; Joshi, Rajendra

2015-01-01

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298–306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K. PMID:26579714

Increased vesicular monoamine transporter binding during early abstinence in human methamphetamine users: Is VMAT2 a stable dopamine neuron biomarker?

PubMed

Boileau, Isabelle; Rusjan, Pablo; Houle, Sylvain; Wilkins, Diana; Tong, Junchao; Selby, Peter; Guttman, Mark; Saint-Cyr, Jean A; Wilson, Alan A; Kish, Stephen J

2008-09-24

Animal data indicate that methamphetamine can damage striatal dopamine terminals. Efforts to document dopamine neuron damage in living brain of methamphetamine users have focused on the binding of [(11)C]dihydrotetrabenazine (DTBZ), a vesicular monoamine transporter (VMAT2) positron emission tomography (PET) radioligand, as a stable dopamine neuron biomarker. Previous PET data report a slight decrease in striatal [(11)C]DTBZ binding in human methamphetamine users after prolonged (mean, 3 years) abstinence, suggesting that the reduction would likely be substantial in early abstinence. We measured striatal VMAT2 binding in 16 recently withdrawn (mean, 19 d; range, 1-90 d) methamphetamine users and in 14 healthy matched-control subjects during a PET scan with (+)[(11)C]DTBZ. Unexpectedly, striatal (+)[(11)C]DTBZ binding was increased in methamphetamine users relative to controls (+22%, caudate; +12%, putamen; +11%, ventral striatum). Increased (+)[(11)C]DTBZ binding in caudate was most marked in methamphetamine users abstinent for 1-3 d (+41%), relative to the 7-21 d (+15%) and >21 d (+9%) groups. Above-normal VMAT2 binding in some drug users suggests that any toxic effect of methamphetamine on dopamine neurons might be masked by an increased (+)[(11)C]DTBZ binding and that VMAT2 radioligand binding might not be, as is generally assumed, a "stable" index of dopamine neuron integrity in vivo. One potential explanation for increased (+)[(11)C]DTBZ binding is that VMAT2 binding is sensitive to changes in vesicular dopamine storage levels, presumably low in drug users. If correct, (+)[(11)C]DTBZ might be a useful imaging probe to correlate changes in brain dopamine stores and behavior in users of methamphetamine.

A Model for Amplification of Hair-Bundle Motion by Cyclical Binding of Ca2+ to Mechanoelectrical-Transduction Channels

NASA Astrophysics Data System (ADS)

Choe, Yong; Magnasco, Marcelo O.; Hudspeth, A. J.

1998-12-01

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca2+ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system's behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca2+ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken's cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

Screening the sequence selectivity of DNA-binding molecules using a gold nanoparticle-based colorimetric approach.

PubMed

Hurst, Sarah J; Han, Min Su; Lytton-Jean, Abigail K R; Mirkin, Chad A

2007-09-15

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4',6-diamidino-2-phenylindone (an AT-specific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex.

UNC-18 Promotes Both the Anterograde Trafficking and Synaptic Function of Syntaxin

PubMed Central

McEwen, Jason M.

2008-01-01

The SM protein UNC-18 has been proposed to regulate several aspects of secretion, including synaptic vesicle docking, priming, and fusion. Here, we show that UNC-18 has a chaperone function in neurons, promoting anterograde transport of the plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein Syntaxin-1. In unc-18 mutants, UNC-64 (Caenorhabditis elegans Syntaxin-1) accumulates in neuronal cell bodies. Colocalization studies and analysis of carbohydrate modifications both suggest that this accumulation occurs in the endoplasmic reticulum. This trafficking defect is specific for UNC-64 Syntaxin-1, because 14 other SNARE proteins and two active zone markers were unaffected. UNC-18 binds to Syntaxin through at least two mechanisms: binding to closed Syntaxin, or to the N terminus of Syntaxin. It is unclear which of these binding modes mediates UNC-18 function in neurons. The chaperone function of UNC-18 was eliminated in double mutants predicted to disrupt both modes of Syntaxin binding, but it was unaffected in single mutants. By contrast, mutations predicted to disrupt UNC-18 binding to the N terminus of Syntaxin caused significant defects in locomotion behavior and responsiveness to cholinesterase inhibitors. Collectively, these results demonstrate the UNC-18 acts as a molecular chaperone for Syntaxin transport in neurons and that the two modes of UNC-18 binding to Syntaxin are involved in different aspects of UNC-18 function. PMID:18596236

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse

PubMed Central

Jacobsen, Jacob P.R.; Plenge, Per; Sachs, Benjamin D.; Pehrson, Alan L.; Cajina, Manuel; Du, Yunzhi; Roberts, Wendy; Rudder, Meghan L.; Dalvi, Prachiti; Robinson, Taylor J.; O’Neill, Sharon P.; Khoo, King S.; Morillo, Connie Sanchez; Zhang, Xiaodong; Caron, Marc G.

2015-01-01

Rationale Escitalopram is a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and antidepressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram’s inhibition hereof. Objectives Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. Methods Recombinant technology; in vivo microdialysis; receptor binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). Results We generated mice expressing either the wild-type human SERT (hSERTWT) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERTALI/VFL+SI/TT). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. Importantly, escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment. Further, escitalopram-induced 5-HTExt elevation was not affected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small, tending to be enhanced by R-citalopram co-administration. Conclusions We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram. Our findings points to mechanisms for R-citalopram antagonism of escitalopram’s antidepressant action other than direct antagonistic binding interactions at the hSERT. PMID:24810106

Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level

PubMed Central

Schvartzman, Mark; Palma, Matteo; Sable, Julia; Abramson, Justin; Hu, Xian; Sheetz, Michael P.; Wind, Shalom J.

2011-01-01

The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least 4 liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures. PMID:21319842

Inferring diffusion dynamics from FCS in heterogeneous nuclear environments.

PubMed

Tsekouras, Konstantinos; Siegel, Amanda P; Day, Richard N; Pressé, Steve

2015-07-07

Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A new approach for bioassays based on frequency- and time-domain measurements of magnetic nanoparticles.

PubMed

Oisjöen, Fredrik; Schneiderman, Justin F; Astalan, Andrea Prieto; Kalabukhov, Alexey; Johansson, Christer; Winkler, Dag

2010-01-15

We demonstrate a one-step wash-free bioassay measurement system capable of tracking biochemical binding events. Our approach combines the high resolution of frequency- and high speed of time-domain measurements in a single device in combination with a fast one-step bioassay. The one-step nature of our magnetic nanoparticle (MNP) based assay reduces the time between sample extraction and quantitative results while mitigating the risks of contamination related to washing steps. Our method also enables tracking of binding events, providing the possibility of, for example, investigation of how chemical/biological environments affect the rate of a binding process or study of the action of certain drugs. We detect specific biological binding events occurring on the surfaces of fluid-suspended MNPs that modify their magnetic relaxation behavior. Herein, we extrapolate a modest sensitivity to analyte of 100 ng/ml with the present setup using our rapid one-step bioassay. More importantly, we determine the size-distributions of the MNP systems with theoretical fits to our data obtained from the two complementary measurement modalities and demonstrate quantitative agreement between them. Copyright 2009 Elsevier B.V. All rights reserved.

Adolescent social defeat alters markers of adult dopaminergic function.

PubMed

Novick, Andrew M; Forster, Gina L; Tejani-Butt, Shanaz M; Watt, Michael J

2011-08-10

Stressful experiences during adolescence can alter the trajectory of neural development and contribute to psychiatric disorders in adulthood. We previously demonstrated that adolescent male rats exposed to repeated social defeat stress show changes in mesocorticolimbic dopamine content both at baseline and in response to amphetamine when tested in adulthood. In the present study we examined whether markers of adult dopamine function are also compromised by adolescent experience of social defeat. Given that the dopamine transporter as well as dopamine D1 receptors act as regulators of psychostimulant action, are stress sensitive and undergo changes during adolescence, quantitative autoradiography was used to measure [(3)H]-GBR12935 binding to the dopamine transporter and [(3)H]-SCH23390 binding to dopamine D1 receptors, respectively. Our results indicate that social defeat during adolescence led to higher dopamine transporter binding in the infralimbic region of the medial prefrontal cortex and higher dopamine D1 receptor binding in the caudate putamen, while other brain regions analyzed were comparable to controls. Thus it appears that social defeat during adolescence causes specific changes to the adult dopamine system, which may contribute to behavioral alterations and increased drug seeking. Copyright © 2011 Elsevier Inc. All rights reserved.

Recognition of Mg²⁺ by a new fluorescent "turn-on" chemosensor based on pyridyl-hydrazono-coumarin.

PubMed

Orrego-Hernández, Jessica; Nuñez-Dallos, Nelson; Portilla, Jaime

2016-05-15

A new fluoroionophore PyHC bearing 2-pyridylhydrazone and 7-hydroxycoumarin moieties for selective detection of Mg(2+) was synthesized and characterized. This chemosensor exhibited "turn-on" fluorescence behavior and was sensitive to Mg(2+) concentrations as low as 105 nmol L(-1) in ethanol-water solution. Detailed spectroscopic studies revealed the binding mode of a 1:1 complex between PyHC and Mg(2+) that leads to a fluorescence enhancement. Copyright © 2016 Elsevier B.V. All rights reserved.

Similar anxiolytic effects of agonists targeting serotonin 5-HT1A or cannabinoid CB receptors on zebrafish behavior in novel environments

PubMed Central

Connors, Kristin A.; Valenti, Theodore W.; Lawless, Kelly; Sackerman, James; Onaivi, Emmanuel S.; Brooks, Bryan W.; Gould, Georgianna G.

2014-01-01

The discovery that selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are present and bioaccumulate in aquatic ecosystems have spurred studies of fish serotonin transporters (SERTs) and changes in SSRI-sensitive behaviors as adverse outcomes relevant for risk assessment. Many SSRIs also act at serotonin 5-HT1A receptors. Since capitolizing on this action may improve treatments of clinical depression and other psychiatric disorders, novel multimodal drugs that agonize 5-HT1A and block SERT were introduced. In mammals both 5-HT1A and CB agonists, such as buspirone and WIN55,212-2, reduce anxious behaviors. Immunological and behavioral evidence suggests that 5-HT1A-like receptors may function similarly in zebrafish (Danio rerio), yet their pharmacological properties are not well characterized. Herein we compared the density of [3H] 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT) binding to 5-HT1A-like sites in the zebrafish brain, to that of simalarly Gαi/o-coupled cannabinoid receptors. [3H] 8-OH-DPAT specific binding was 176 ± 8, 275 ± 32, and 230 ± 36 fmol/mg protein in the hypothalamus, optic tectum, and telencephalon. [3H] WIN55,212-2 binding density was higher in those same brain regions at 6 ± 0.3, 5.5 ± 0.4 and 7.3 ± 0.3 pm/mg protein. The aquatic light-dark plus maze was used to examine behavioral effects of 5-HT1A and CB receptor agonists on zebrafish novelty-based anxiety. With acute exposure to the 5-HT1A partial-agonist buspirone (50 mg/L), or dietary exposure to WIN55,212-2 (7 μg/week) zebrafish spent more time in and/or entered white arms more often than controls (p < 0.05). Acute exposure to WIN55,212-2 at 0.5-50 mg/L, reduced mobility. These behavioral findings suggest that azipirones, like cannabinoid agonists, have anxiolytic and/or sedative properties on fish in novel environments. These observations highlight the need to consider potential ecological risks of azapirones and multimodal antidepressants in the future. PMID:24411165

Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.

1991-05-01

We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broadmore » distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.« less

Postnatal haloperidol eliminates the deficit in circling behavior produced by prenatal exposure to the same drug.

PubMed

Wolansky, Marcelo Javier; Soiza-Reilly, Mariano; Fossati, Mariana; Azcurra, Julio Marcos

2004-01-01

Up to 35% of pregnant women take psychotropic drugs at least once during gestation [Austin and Mitchell, 1998]. From concurrent animal and human evidence, it has been proposed that exposure to several psychoactive medications in utero or during lactation increases the risk for permanent brain disorders. Present preventive or therapy practices applied on humans for this type of long-lasting behavioral alterations are mainly based on empirical results. Here, we test an experimental approach designed to counteract a circling performance deficit that appears in Sprague-Dawley rats at puberty on exposure to the dopaminergic blocker haloperidol (HAL) during gestation [J.L. Brusés, J.M. Azcurra, The circling training: A behavioral paradigm for functional teratology testing, in: P.M. Conn (Ed.), Paradigms for the study of behavior, Acad. Press, New York, 1993, pp. 166-179. Method Neurosci. 14]. Gestational exposure to HAL (GD 5-18, 2.5 mg/kg/day ip) induced the expected circling activity decrease in the offspring at the fifth week of life. When prenatal exposure to HAL was continued through lactation (PD5-21, 1.5 mg/kg/day ip), rats otherwise showed a control-like circling performance. No difference was yet found between lactation-only, HAL-exposed pups and saline (SAL)-treated controls (n=8 each group). We further performed saturating (3H)-spiroperidol (SPI) binding assays on striatal P2 membrane fractions 2 months later. The dopamine-type D2-specific binding results suggested that above circling behavior findings could be partially explained by enduring HAL-induced neurochemical changes. The role of critical periods of sensitivity as transient windows for opportunistic therapies for behavioral teratology is discussed. Copyright 2004 Elsevier Inc.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

Brain mu-opioid receptor binding predicts treatment outcome in cocaine-abusing outpatients

PubMed Central

Ghitza, Udi E.; Preston, Kenzie L.; Epstein, David H.; Kuwabara, Hiroto; Endres, Christopher J.; Bencherif, Badreddine; Boyd, Susan J.; Copersino, Marc L.; Frost, J. James; Gorelick, David A.

2010-01-01

Background Cocaine users not seeking treatment have increased regional brain mu-opioid receptor (mOR) binding that correlates with cocaine craving and tendency to relapse. In cocaine-abusing outpatients in treatment, the relationship of mOR binding and treatment outcome is unknown. Methods We determined whether regional brain mOR binding before treatment correlates with outcome and compared it to standard clinical predictors of outcome. Twenty-five individuals seeking outpatient treatment for cocaine abuse or dependence (DSM-IV) received up to 12 weeks of cognitive-behavioral therapy and cocaine-abstinence reinforcement whereby each cocaine-free urine was reinforced with vouchers redeemable for goods. Regional brain mOR binding was measured before treatment using positron emission tomography (PET) with [11C] carfentanil (a selective mOR agonist). Main outcome measures were: 1) overall percentage of urines positive for cocaine during first month of treatment, 2) longest duration (weeks) of abstinence from cocaine during treatment, all verified by urine toxicology. Results Elevated mOR binding in the medial frontal and middle frontal gyri before treatment correlated with greater cocaine use during treatment. Elevated mOR binding in the anterior cingulate, medial frontal, middle frontal, middle temporal, and sub-lobar insular gyri correlated with shorter duration of cocaine abstinence during treatment. Regional mOR binding contributed significant predictive power for treatment outcome beyond that of standard clinical variables such as baseline drug and alcohol use. Conclusions Elevated mOR binding in brain regions associated with reward sensitivity is a significant independent predictor of treatment outcome in cocaine-abusing outpatients, suggesting a key role for the brain endogenous opioid system in cocaine addiction. PMID:20579973

Of pheromones and kairomones: what receptors mediate innate emotional responses?

PubMed

Fortes-Marco, Lluis; Lanuza, Enrique; Martinez-Garcia, Fernando

2013-09-01

Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation. Copyright © 2013 Wiley Periodicals, Inc.

Graphene oxide-based optical biosensor functionalized with peptides for explosive detection.

PubMed

Zhang, Qian; Zhang, Diming; Lu, Yanli; Yao, Yao; Li, Shuang; Liu, Qingjun

2015-06-15

A label-free optical biosensor was constructed with biofunctionalized graphene oxide (GO) for specific detection of 2,4,6-trinitrotoluene (TNT). By chemically binding TNT-specific peptides with GO, the biosensor gained unique optoelectronic properties and high biological sensitivity, with transducing bimolecular bonding into optical signals. Through UV absorption detection, increasing absorbance responses could be observed in presence of TNT at different concentrations, as low as 4.40×10(-9) mM, and showed dose-dependence and stable behavior. Specific responses of the biosensor were verified with the corporation of 2,6-dinitrotoluene (DNT), which had similar molecular structure to TNT. Thus, with high sensitivity and selectivity, the biosensor provided a convenient approach for detection of explosives as miniaturizing and integrating devices. Copyright © 2015 Elsevier B.V. All rights reserved.

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

PubMed

Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

2015-02-15

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Sarcoptes scabiei Tropomyosin and Paramyosin: Immunoreactive Allergens in Scabies.

PubMed

Naz, Shumaila; Desclozeaux, Marion; Mounsey, Kate E; Chaudhry, Farhana Riaz; Walton, Shelley F

2017-09-01

Scabies is a human skin disease due to the burrowing ectoparasite Sarcoptes scabiei var. hominis resulting in intense itching and inflammation and manifesting as a skin allergy. Because of insufficient mite material and lack of in vitro propagation system for antigen preparation, scabies is a challenging disease to develop serological diagnostics. For allergen characterization, full-length S. scabiei tropomyosin (Sar s 10) was cloned, expressed in pET-15b, and assessed for reactivity with IgE antibodies from human sera. IgE binding was observed to Sar s 10 with sera collected from subjects with ordinary scabies, house dust mite (HDM)-positive and naive subjects and a diagnostic sensitivity of < 30% was observed. S. scabiei paramyosin (Sar s 11) was cloned, and expressed in pET-28a in three overlapping fragments designated Sspara1, Sspara2, and Sspara3. IgE and IgG binding was observed to Sspara2 and Sspara3 antigens with sera collected from ordinary scabies, and HDM-positive subjects, but no binding was observed with sera collected from naive subjects. Sspara2 displayed excellent diagnostic potential with 98% sensitivity and 90% specificity observed for IgE binding and 70% sensitivity for IgG. In contrast, the diagnostic sensitivity of Sspara3 was 84% for IgE binding and 40% for IgG binding. In combination, Sspara2 and Sspara3 provided an IgE sensitivity of 94%. This study shows that IgE binding to Sspara2 and Sspara3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice. The developed enzyme-linked immunosorbent assay helps direct future development of a specific diagnostic tool for scabies.

Narp Deletion Blocks Extinction of Morphine Place Preference Conditioning

PubMed Central

Crombag, Hans S; Dickson, Mercy; Dinenna, Megan; Johnson, Alexander W; Perin, Mark S; Holland, Peter C; Baraban, Jay M; Reti, Irving M

2008-01-01

As drug abuse can be viewed as a maladaptive form of neuronal plasticity, attention has focused on defining the synaptic plasticity mechanisms that mediate the long-term effects of these drugs. As Narp is secreted at synaptic sites and binds to the extracellular surface of AMPA receptors, it has been implicated in mediating enduring forms of synaptic plasticity. Accordingly, to assess its potential role in the long-lasting behavioral effects of drugs of abuse, we have investigated the impact of Narp deletion on sustained behavioral responses elicited by repeated morphine administration. Narp knockout mice display normal locomotor sensitization and conditioned place preference, but are markedly resistant to extinction of place preference. Thus, these findings indicate that Narp plays a selective role in extinction, possibly by its effects on AMPA receptor trafficking. PMID:18536700

A density functional theory study of the correlation between analyte basicity, ZnPc adsorption strength, and sensor response.

PubMed

Tran, N L; Bohrer, F I; Trogler, W C; Kummel, A C

2009-05-28

Density functional theory (DFT) simulations were used to determine the binding strength of 12 electron-donating analytes to the zinc metal center of a zinc phthalocyanine molecule (ZnPc monomer). The analyte binding strengths were compared to the analytes' enthalpies of complex formation with boron trifluoride (BF(3)), which is a direct measure of their electron donating ability or Lewis basicity. With the exception of the most basic analyte investigated, the ZnPc binding energies were found to correlate linearly with analyte basicities. Based on natural population analysis calculations, analyte complexation to the Zn metal of the ZnPc monomer resulted in limited charge transfer from the analyte to the ZnPc molecule, which increased with analyte-ZnPc binding energy. The experimental analyte sensitivities from chemiresistor ZnPc sensor data were proportional to an exponential of the binding energies from DFT calculations consistent with sensitivity being proportional to analyte coverage and binding strength. The good correlation observed suggests DFT is a reliable method for the prediction of chemiresistor metallophthalocyanine binding strengths and response sensitivities.

G3139, an Anti-Bcl-2 Antisense Oligomer that Binds Heparin-Binding Growth Factors and Collagen I, Alters In Vitro Endothelial Cell Growth and Tubular Morphogenesis

PubMed Central

Stein, C.A.; Wu, SiJian; Voskresenskiy, Anatoliy M.; Zhou, Jin-Feng; Shin, Joongho; Miller, Paul; Souleimanian, Naira; Benimetskaya, Luba

2009-01-01

Purpose We examined the effects of G3139 on the interaction of heparin-binding proteins (e.g., FGF2 and collagen I) with endothelial cells. G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global Phase III trial in advanced melanoma (GM301) has evaluted G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood, as it is unlikely that Bcl-2 silencing is the sole mechanism for chemo-sensitization in melanoma cells. Experimental Design The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high affinity cell surface receptors, and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and HUVEC cells were determined. Results G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and from proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in 3D collagen gels, increases the mitogenesis of HUVEC cells similarly, and induces vessel sprouts in the rat aortic ring model. Conclusions G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with LDH observed clinically. PMID:19351753

Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition.

PubMed

Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; De Biasi, Federico; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

2017-07-13

The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the monolayer itself and with the solvent. We further describe how the nature and plasticity of these interactions modulate nanoparticle-based chemosensing. Importantly, we found that self-organization of coating thiols can induce the formation of binding pockets in AuNPs. These transient cavities can accommodate small molecules, mimicking protein-ligand recognition, which could explain the selectivity and sensitivity observed for different organic analytes in NMR chemosensing experiments. Thus, our findings advocate for the rational design of tailored coating groups to form specific recognition binding sites on monolayer-protected AuNPs.

A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor.

PubMed

Barchad-Avitzur, Ofra; Priest, Michael F; Dekel, Noa; Bezanilla, Francisco; Parnas, Hanna; Ben-Chaim, Yair

2016-10-04

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Electrochemical surface-enhanced Raman scattering measurement on ligand capped PbS quantum dots at gap of Au nanodimer

NASA Astrophysics Data System (ADS)

Li, Xiaowei; Minamimoto, Hiro; Murakoshi, Kei

2018-05-01

The vibrational characteristics of ligand-capped lead sulfide (PbS) quantum dots (QDs) were clarified via electrochemical surface-enhanced Raman spectroscopy (EC-SERS) using a hybridized system of gold (Au) nanodimers and PbS QDs under electrochemical potential control. Enhanced electromagnetic field caused by the coupling of QDs with plasmonic Au nanodimers allowed the characteristic behavior of the ligand oleic acid (OA) on the PbS QD surface to be detected under electrochemical potential control. Binding modes between the QDs and OA molecules were characterized using synchronous two-dimensional correlation spectra at distinct electrochemical potentials, confirming that the bidentate bridging mode was probably the most stable mode even under relatively negative potential polarization. Changes in binding modes and molecular orientations resulted in fluctuations in EC-SERS spectra. The present observations strongly recommend the validity of the QD-plasmonic nanostructure coupled system for sensitive molecular detection via EC-SERS.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

PubMed

Schröter, Christian; Günther, Ralf; Rhiel, Laura; Becker, Stefan; Toleikis, Lars; Doerner, Achim; Becker, Janine; Schönemann, Andreas; Nasu, Daichi; Neuteboom, Berend; Kolmar, Harald; Hock, Björn

2015-01-01

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

PubMed

Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

2017-08-01

Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Opiate receptor binding in the brain of the seizure sensitive Mongolian gerbil (Meriones unguiculatus).

PubMed

Lee, R J; Olsen, R W; Lomax, P; McCabe, R T; Wamsley, J K

1984-12-01

Opiate receptor binding was studied in seizure sensitive (SS) and seizure resistant (SR) strains of the Mongolian gerbil. Cryostat sections of the brain were labeled with [3H]-dihydromorphine, subjected to autoradiography and analysed by microdensitometry. SS gerbils, prior to seizure induction, demonstrated overall greater brain opiate binding when compared to SR animals. Immediately following a seizure, binding in the interpeduncular nucleus fell to levels found in SR animals. The increased opiate binding in the SS (pre-seizure) compared to SR gerbils could reflect a deficit of endogenous ligand which could underlie the seizure diathesis in the gerbil.

Precise identification and manipulation of adsorption geometry of donor-π-acceptor dye on nanocrystalline TiO₂ films for improved photovoltaics.

PubMed

Zhang, Fan; Ma, Wei; Jiao, Yang; Wang, Jingchuan; Shan, Xinyan; Li, Hui; Lu, Xinghua; Meng, Sheng

2014-12-24

Adsorption geometry of dye molecules on nanocrystalline TiO2 plays a central role in dye-sensitized solar cells, enabling effective sunlight absorption, fast electron injection, optimized interface band offsets, and stable photovoltaic performance. However, precise determination of dye binding geometry and proportion has been challenging due to complexity and sensitivity at interfaces. Here employing combined vibrational spectrometry and density functional calculations, we identify typical adsorption configurations of widely adopted cyanoacrylic donor-π bridge-acceptor dyes on nanocrystalline TiO2. Binding mode switching from bidentate bridging to hydrogen-bonded monodentate configuration with Ti-N bonding has been observed when dye-sensitizing solution becomes more basic. Raman and infrared spectroscopy measurements confirm this configuration switch and determine quantitatively the proportion of competing binding geometries, with vibration peaks assigned using density functional theory calculations. We further found that the proportion of dye-binding configurations can be manipulated by adjusting pH value of dye-sensitizing solutions. Controlling molecular adsorption density and configurations led to enhanced energy conversion efficiency from 2.4% to 6.1% for the fabricated dye-sensitized solar cells, providing a simple method to improve photovoltaic performance by suppressing unfavorable binding configurations in solar cell applications.

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

PubMed

Bertsch, M; Mayburd, A L; Kassner, R J

2003-02-15

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

PubMed Central

Wang, Jing; Tang, Shan; Wan, Zhengpeng; Gao, Yiren; Cao, Yiyun; Yi, Junyang; Si, Yanyan; Zhang, Haowen; Liu, Lei; Liu, Wanli

2016-01-01

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens. PMID:26764382

Highly Sensitive Aluminium(III) Ion Sensor Based on a Self-assembled Monolayer on a Gold Nanoparticles Modified Screen-printed Carbon Electrode.

PubMed

See, Wong Pooi; Heng, Lee Yook; Nathan, Sheila

2015-01-01

A new approach for the development of a highly sensitive aluminium(III) ion sensor via the preconcentration of aluminium(III) ion with a self-assembled monolayer on a gold nanoparticles modified screen-printed carbon electrode and current mediation by potassium ferricyanide redox behavior during aluminium(III) ion binding has been attempted. A monolayer of mercaptosuccinic acid served as an effective complexation ligand for the preconcentration of trace aluminium; this led to an enhancement of aluminium(III) ion capture and thus improved the sensitivity of the sensor with a detection limit of down to the ppb level. Under the optimum experimental conditions, the sensor exhibited a wide linear dynamic range from 0.041 to 12.4 μM. The lower detection limit of the developed sensor was 0.037 μM (8.90 ppb) using a 10 min preconcentration time. The sensor showed excellent selectivity towards aluminium(III) ion over other interference ions.

Basis for changes in the auxin-sensitivity of Avena sativa (oat) leaf-sheath pulvini during the gravitropic response

NASA Technical Reports Server (NTRS)

Kim, D.; Kaufman, P. B.

1995-01-01

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

The elusive permeability barriers and binding sites for proflavine in Escherichia coli.

PubMed

Gravelle, M J; Mehta, B M; Kushner, D J

1972-06-01

Cells of proflavine-sensitive and -resistant Escherichia coli strains were altered in different ways, and the proflavine binding of the changed material was studied. Spheroplasts prepared from sensitive and resistant cells bound similar amounts of proflavine at saturation, whether or not they were osmotically protected by 10% sucrose. Intact cells bound approximately the same amounts of proflavine as spheroplasts. On addition of glucose, osmotically protected resistant but not sensitive spheroplasts released proflavine; unprotected spheroplasts did not release bound proflavine. Thus, osmotically protected membranes are not required for proflavine binding (a passive process) but are required for proflavine release (an active process). The presence of sucrose reduced proflavine binding by resistant cells. Adding glucose to cells in 20% sucrose did not cause a release of residual proflavine, though glucose caused a release of proflavine from cells suspended in 0 or 10% sucrose. On treatment of heated cells or ruptured spheroplasts with nucleases and Pronase, practically all nucleic acids were removed. Proflavine-binding ability of such preparations fell by only 30 to 50%. Washing heated cells with ethanol did not reduce their proflavine-binding ability. There appear to be important binding sites in cells aside from nucleic acids.

The Elusive Permeability Barriers and Binding Sites for Proflavine in Escherichia coli

PubMed Central

Gravelle, M. Joan; Mehta, B. M.; Kushner, D. J.

1972-01-01

Cells of proflavine-sensitive and -resistant Escherichia coli strains were altered in different ways, and the proflavine binding of the changed material was studied. Spheroplasts prepared from sensitive and resistant cells bound similar amounts of proflavine at saturation, whether or not they were osmotically protected by 10% sucrose. Intact cells bound approximately the same amounts of proflavine as spheroplasts. On addition of glucose, osmotically protected resistant but not sensitive spheroplasts released proflavine; unprotected spheroplasts did not release bound proflavine. Thus, osmotically protected membranes are not required for proflavine binding (a passive process) but are required for proflavine release (an active process). The presence of sucrose reduced proflavine binding by resistant cells. Adding glucose to cells in 20% sucrose did not cause a release of residual proflavine, though glucose caused a release of proflavine from cells suspended in 0 or 10% sucrose. On treatment of heated cells or ruptured spheroplasts with nucleases and Pronase, practically all nucleic acids were removed. Proflavine-binding ability of such preparations fell by only 30 to 50%. Washing heated cells with ethanol did not reduce their proflavine-binding ability. There appear to be important binding sites in cells aside from nucleic acids. PMID:4618456

Pyrethroid receptor in the insect Na sup + channel: Alteration of its properties in pyrethroid-resistant flies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pauron, D.; Barhanin, J.; Amichot, M.

1989-02-21

Resistance to insecticides is a major problem in agriculture. ({sup 3}H)Saxitoxin binding experiments have shown that pyrethroid-sensitive and pyrethroid-resistant flies have the same amount of Na{sup +} channel protein in their brain membranes. Also, although flies are resistant to pyrethroids, they remain as sensitive to batrachotoxin, which is another type of Na{sup +} channel activators, as pyrethroid-sensitive flies. Pyrethroid binding sites have been characterized by use of the properties of pyrethroids to increase the specific ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate binding component. K{sub 0.5} values for association of pyrethroids at the Na{sup +} channel of pyrethroid-sensitive flies are in the rangemore » of 0.15-0.25 {mu}M. Conversely, pyrethroids do not produce a significant increase of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate binding in pyrethroid-resistant flies even at high concentrations of the insecticide. It is concluded that linkage between pyrethroid and batrachotoxin binding sites is altered in the pyrethroid-resistant fly strains. This alteration is probably due to a drastically decreased affinity of the Na{sup +} channel for pyrethroids.« less

Ginseng and the hypothalamic-pituitary control of stress.

PubMed

Fulder, S J

1981-01-01

There are a group of so-called tonic remedies in Far Eastern medicine which are traditionally viewed as harmonizing or adjustive. Ginseng and eleutherococcus are the best known, and there is evidence that they increase arousal, stamina and stress resistance. We have attempted to explore the relationship between the behavioral and the stress effects, and to relate this to traditional concepts. In one series of experiments mice were given ginseng throughout their lifespan. At intervals their behavior response to mild stress was examined and found to be exaggerated compared to controls without ginseng. However, normal ambulatory behavior in the absence of stress was unaffected. A second series of experiments indicated that the binding of corticosteroid to certain brain regions was increased in adrenalectomized rats given ginseng saponin, compared to saline treated controls. This can be interpreted as a result of an increase in hypothalamic-pituitary-adrenal sensitivity caused by ginseng saponin. This is in accord with traditional concepts of the use of these remedies.

On the Role of Water Models in Quantifying the Binding Free Energy of Highly Conserved Water Molecules in Proteins: The Case of Concanavalin A.

PubMed

Fadda, Elisa; Woods, Robert J

2011-10-11

The ability of ligands to displace conserved water molecules in protein binding sites is of significant interest in drug design and is particularly pertinent in the case of glycomimetic drugs. This concept was explored in previous work [ Clarke et al. J. Am. Chem. Soc. 2001 , 123 , 12238 - 12247 and Kadirvelraj et al. J. Am. Chem. Soc. 2008 , 130 , 16933 - 16942 ] for a highly conserved water molecule located in the binding site of the prototypic carbohydrate-binding protein Concanavalin A (Con A). A synthetic ligand was designed with the aim of displacing such water. While the synthetic ligand bound to Con A in an analogous manner to that of the natural ligand, crystallographic analysis demonstrated that it did not displace the conserved water. In order to quantify the affinity of this particular water for the Con A surface, we report here the calculated standard binding free energy for this water in both ligand-bound and free Con A, employing three popular water models: TIP3P, TIP4P, and TIP5P. Although each model was developed to perform well in simulations of bulk-phase water, the computed binding energies for the isolated water molecule displayed a high sensitivity to the model. Both molecular dynamics simulation and free energy results indicate that the choice of water model may greatly influence the characterization of surface water molecules as conserved (TIP5P) or not (TIP3P) in protein binding sites, an observation of considerable significance to rational drug design. Structural and theoretical aspects at the basis of the different behaviors are identified and discussed.

Interactions of carbon nanotubes and/or graphene with manganese peroxidase during biodegradation of endocrine disruptors and triclosan.

PubMed

Chen, Ming; Zeng, Guangming; Lai, Cui; Zhang, Chang; Xu, Piao; Yan, Min; Xiong, Weiping

2017-10-01

Molecular-level biodegradation processes of bisphenol A (BPA), nonylphenol (NP) and triclosan (TCS) mediated by manganese peroxidase (MnP) were investigated with and without single-walled carbon nanotube (SWCNT) and/or graphene (GRA). Although the incorporation of SWCNT, GRA or their combination (SWCNT+GRA) did not break up the complexes composed of manganese peroxidase (MnP) and these substrates, they had different effects on the native contacts between the substrates and MnP. GRA tended to decrease the overall stability of the binding between MnP and its substrates. SWCNT or SWCNT+GRA generally had a minor impact on the mean binding energy between MnP and its substrates. We detected some sensitive residues from MnP that were dramatically disturbed by the GRA, SWCNT or SWCNT+GRA. Nanomaterials changed the number and behavior of water molecules adjacent to both MnP and its substrates, which was not due to the destruction of H-bond network formed by sensitive regions and water molecules. The present results are useful for understanding the molecular basis of pollutant biodegradation affected by the nanomaterials in the environment, and are also helpful in assessing the risks of these materials to the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions-Molecular Imprinting and QCMs †.

PubMed

Mujahid, Adnan; Mustafa, Ghulam; Dickert, Franz L

2018-06-01

Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs). The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM) resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

PubMed

Tillotson, Benjamin J; Goulatis, Loukas I; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V

2015-01-01

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

PubMed Central

Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

2015-01-01

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

An X-ray photoelectron spectroscopy study of BF3 adsorption on positively and negatively poled LiNbO3 (0001)

NASA Astrophysics Data System (ADS)

Herdiech, M. W.; Mönig, H.; Altman, E. I.

2014-08-01

Adsorption of the strong Lewis acid BF3 was investigated to probe the sensitivity of the Lewis basicity of surface oxygens on LiNbO3 (0001) to the ferroelectric polarization direction. Adsorption and desorption were characterized by using X-ray photoelectron spectroscopy (XPS) to monitor the intensity and binding energy of the F 1s core level as a function of BF3 exposure and temperature. The results indicate that both BF3 uptake and desorption are very similar on the positively and negatively poled surfaces. In particular, BF3 only weakly adsorbs with the majority of the adsorbed BF3 desorbing below 200 K. Despite the similarities in the uptake and desorption behavior, the binding energy of the F 1s peak relative to the substrate Nb 3d5/2 peak was sensitive to the polarization direction, with the F 1s peak occurring at a binding energy up to 0.3 eV lower on positively poled than negatively poled LiNbO3 for equivalent BF3 exposures. Rather than reflecting a difference in bonding to the surface, however, this shift could be associated with oppositely oriented dipoles at the positively and negatively poled surfaces creating opposite band offsets between the adsorbate and the substrate. A similar effect was observed with lead zirconate titanate thin films where the Pb 4f XPS peak position changes as a function of temperature as a result of the pyroelectric effect which changes the magnitude of the surface and interface dipoles.

Elevation of naloxone-sensitive /sup 3/H-dihydromorphine binding in hippocampal formation of genetically epilepsy-prone rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savage, D.D.; Mills, S.A.; Jobe, P.C.

1988-01-01

/sup 3/H-Dihydromorphine (DHM) binding sites were measured in the brain of non-epileptic control and GEPR rats using in vitro autoradiographic techniques. The number of naloxone-sensitive /sup 3/H-DHM binding sites was increased 38-57% in the pyramidal cell layer of ventral hippocampal CA/sub 3/ and CA/sub 1/ of GEPR-3 and GEPR-9 rats compared to non-epileptic controls. No significant differences in /sup 3/H-DHM binding were observed in dorsal hippocampal formation, lateral entorhinal cortex, lateral geniculate or cerebellum. The results suggest that an increase in the number of opioid receptors in ventral hippocampus of GEPR rats may be one factor contributing to the enhancedmore » sensitivity of GEPR-9 rats to the proconvulsant effects of morphine.« less

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

PubMed

Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

Age and sex differences in oxytocin and vasopressin V1a receptor binding densities in the rat brain: focus on the social decision-making network.

PubMed

Smith, Caroline J W; Poehlmann, Max L; Li, Sara; Ratnaseelan, Aarane M; Bredewold, Remco; Veenema, Alexa H

2017-03-01

Oxytocin (OT) and vasopressin (AVP) regulate various social behaviors via activation of the OT receptor (OTR) and the AVP V1a receptor (V1aR) in the brain. Social behavior often differs across development and between the sexes, yet our understanding of age and sex differences in brain OTR and V1aR binding remains incomplete. Here, we provide an extensive analysis of OTR and V1aR binding density throughout the brain in juvenile and adult male and female rats, with a focus on regions within the social decision-making network. OTR and V1aR binding density were higher in juveniles than in adults in regions associated with reward and socio-spatial memory and higher in adults than in juveniles in key regions of the social decision-making network and in cortical regions. We discuss possible implications of these shifts in OTR and V1aR binding density for the age-specific regulation of social behavior. Furthermore, sex differences in OTR and V1aR binding density were less numerous than age differences. The direction of these sex differences was region-specific for OTR but consistently higher in females than in males for V1aR. Finally, almost all sex differences in OTR and V1aR binding density were already present in juveniles and occurred in regions with denser binding in adults compared to juveniles. Possible implications of these sex differences for the sex-specific regulation of behavior, as well potential underlying mechanisms, are discussed. Overall, these findings provide an important framework for testing age- and sex-specific roles of OTR and V1aR in the regulation of social behavior.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

PubMed

Zhang, Zhongqiang; Gao, Bingsi; Zhao, Chengjiang; Long, Cassandra; Qi, Haizhi; Ezzelarab, Mohamed; Cooper, David Kc; Hara, Hidetaka

2017-07-01

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 μL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 μL to 20 μL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nuclear localization of the DNA repair scaffold XRCC1: Uncovering the functional role of a bipartite NLS

DOE PAGES

Kirby, Thomas W.; Gassman, Natalie R.; Smith, Cassandra E.; ...

2015-08-25

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs ofmore » the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin β binding domain. This behavior gives a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.« less

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steiner, Andrew W.; Lattimer, James M.; Brown, Edward F.

We investigate constraints on neutron star structure arising from the assumptions that neutron stars have crusts, that recent calculations of pure neutron matter limit the equation of state of neutron star matter near the nuclear saturation density, that the high-density equation of state is limited by causality and the largest high-accuracy neutron star mass measurement, and that general relativity is the correct theory of gravity. We explore the role of prior assumptions by considering two classes of equation of state models. In a first, the intermediate- and high-density behavior of the equation of state is parameterized by piecewise polytropes. Inmore » the second class, the high-density behavior of the equation of state is parameterized by piecewise continuous line segments. The smallest density at which high-density matter appears is varied in order to allow for strong phase transitions above the nuclear saturation density. We critically examine correlations among the pressure of matter, radii, maximum masses, the binding energy, the moment of inertia, and the tidal deformability, paying special attention to the sensitivity of these correlations to prior assumptions about the equation of state. It is possible to constrain the radii of 1.4 solar mass neutron stars to be larger than 10 km, even without consideration of additional astrophysical observations, for example, those from photospheric radius expansion bursts or quiescent low-mass X-ray binaries. We are able to improve the accuracy of known correlations between the moment of inertia and compactness as well as the binding energy and compactness. Furthermore, we also demonstrate the existence of a correlation between the neutron star binding energy and the moment of inertia.« less

Spatially Resolved Sensitivity of Single-Particle Plasmon Sensors

PubMed Central

2018-01-01

The high sensitivity of localized surface plasmon resonance sensors to the local refractive index allows for the detection of single-molecule binding events. Though binding events of single objects can be detected by their induced plasmon shift, the broad distribution of observed shifts remains poorly understood. Here, we perform a single-particle study wherein single nanospheres bind to a gold nanorod, and relate the observed plasmon shift to the binding location using correlative microscopy. To achieve this we combine atomic force microscopy to determine the binding location, and single-particle spectroscopy to determine the corresponding plasmon shift. As expected, we find a larger plasmon shift for nanospheres binding at the tip of a rod compared to its sides, in good agreement with numerical calculations. However, we also find a broad distribution of shifts even for spheres that were bound at a similar location to the nanorod. Our correlative approach allows us to disentangle effects of nanoparticle dimensions and binding location, and by comparison to numerical calculations we find that the biggest contributor to this observed spread is the dispersion in nanosphere diameter. These experiments provide insight into the spatial sensitivity and signal-heterogeneity of single-particle plasmon sensors and provides a framework for signal interpretation in sensing applications. PMID:29520315

Specific deficit of colour-colour short-term memory binding in sporadic and familial Alzheimer's disease.

PubMed

Parra, Mario A; Sala, Sergio Della; Abrahams, Sharon; Logie, Robert H; Méndez, Luis Guillermo; Lopera, Francisco

2011-06-01

Short-term memory binding of visual features which are processed across different dimensions (shape-colour) is impaired in sporadic Alzheimer's disease, familial Alzheimer's disease, and in asymptomatic carriers of familial Alzheimer's disease. This study investigated whether Alzheimer's disease also impacts on within-dimension binding processes. The study specifically explored whether visual short-term memory binding of features of the same type (colour-colour) is sensitive to Alzheimer's disease. We used a neuropsychological battery and a short-term memory binding task to assess patients with sporadic Alzheimer's disease (Experiment 1), familial Alzheimer's disease (Experiment 2) due to the mutation E280A of the Presenilin-1 gene and asymptomatic carriers of the mutation. The binding task assessed change detection within arrays of unicoloured objects (Colour Only) or bicoloured objects the colours of which had to be remembered separately (Unbound Colours) or together (Bound Colours). Performance on the Bound Colours condition (1) explained the largest proportion of variance between patients (sporadic and familial Alzheimer's disease), (2) combined more sensitivity and specificity for the disease than other more traditional neuropsychological tasks, (3) identified asymptomatic carriers of the mutation even when traditional neuropsychological measures and other measures of short-term memory did not and, (4) contrary to shape-colour binding, correlated with measures of hippocampal functions. Colour-colour binding and shape-colour binding both appear to be sensitive to AD even though they seem to rely on different brain mechanisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

Vicilin allergens of peanut and tree nuts (walnut, hazelnut and cashew nut) share structurally related IgE-binding epitopes.

PubMed

Barre, Annick; Sordet, Camille; Culerrier, Raphaël; Rancé, Fabienne; Didier, Alain; Rougé, Pierre

2008-03-01

Surface-exposed IgE-binding epitopes of close overall conformation were characterized on the molecular surface of three-dimensional models built for the vicilin allergens of peanut (Ara h 1), walnut (Jug r 2), hazelnut (Cor a 11) and cashew nut (Ana o 1). They correspond to linear stretches of conserved amino acid sequences mainly located along the C-terminus of the polypeptide chains. A glyco-epitope corresponding to an exposed N-glycosylation site could also interfere with the IgE-binding epitopes. All these epitopic regions should participate in the IgE-binding cross-reactivity commonly reported between tree nuts or between peanut and some tree nuts in sensitized individuals. Owing to this epitopic community which constitutes a risk of cross-sensitization, the avoidance or a restricted consumption of other tree nuts should be recommended to peanut-sensitized individuals.

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

NASA Astrophysics Data System (ADS)

Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

PubMed Central

Taft, W C; DeLorenzo, R J

1984-01-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

Membrane Curvature Sensing by Amphipathic Helices

PubMed Central

Jensen, Martin Borch; Bhatia, Vikram Kjøller; Jao, Christine C.; Rasmussen, Jakob Ewald; Pedersen, Søren L.; Jensen, Knud J.; Langen, Ralf; Stamou, Dimitrios

2011-01-01

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues. PMID:21953452

Peripheral gustatory processing of sweet stimuli by golden hamsters.

PubMed

Frank, Marion E; Formaker, Bradley K; Hettinger, Thomas P

2005-07-15

Behaviors and taste-nerve responses to bitter stimuli are linked to compounds that bind T2 receptors expressed in one subset of taste-bud receptor cells (TRCs); and behavioral and neural responses to sweet stimuli are linked to chemical compounds that bind a T1 receptor expressed in a different TRC subset. Neural and behavioral responses to bitter-sweet mixtures, however, complicate the ostensible bitter and sweet labeled lines. In the golden hamster, Mesocricetus auratus, quinine hydrochloride, the bitter prototype, suppresses chorda tympani (CT) nerve responses to the sweet prototype: sucrose. This bitter-sweet inhibition was tested with concentration series of sucrose and dulcin, a hydrophobic synthetic sweetener that hamsters behaviorally cross-generalize with sucrose. Dulcin, sucrose and other sweeteners activate one subset of CT fibers: S neurons; whereas, quinine activates a separate subset of CT fibers: E neurons. Whole-nerve and S-neuron CT responses to a sweetener concentration series, mixed with 0, 1, 3 and 10 mM quinine, were measured for 0-2.5 s transient and/or 2.6-10 s steady-state response periods. Ten-sec total single-fiber records, aligned at response onset, were averaged for 100 ms bins to identify response oscillations. Quinine inhibition of dulcin and sucrose responses was identical. Each log molar increment in quinine resulted in equivalent declines in response to either sweetener. Furthermore, sucrose response decrements paralleled response increments in quinine-sensitive CT neurons to the same quinine increases. A 1.43 Hz bursting rhythm to the sweeteners was unchanged by quinine inhibition or decreases in sweetener concentration. Taste-bud processing, possibly between-cell inhibition and within-cell negative feedback, must modify signals initiated by T1 receptors before they are transmitted to the brain.

Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.

PubMed

Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

2013-06-18

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Thin Hydrogel Films for Optical Biosensor Applications

PubMed Central

Mateescu, Anca; Wang, Yi; Dostalek, Jakub; Jonas, Ulrich

2012-01-01

Hydrogel materials consisting of water-swollen polymer networks exhibit a large number of specific properties highly attractive for a variety of optical biosensor applications. This properties profile embraces the aqueous swelling medium as the basis of biocompatibility, non-fouling behavior, and being not cell toxic, while providing high optical quality and transparency. The present review focuses on some of the most interesting aspects of surface-attached hydrogel films as active binding matrices in optical biosensors based on surface plasmon resonance and optical waveguide mode spectroscopy. In particular, the chemical nature, specific properties, and applications of such hydrogel surface architectures for highly sensitive affinity biosensors based on evanescent wave optics are discussed. The specific class of responsive hydrogel systems, which can change their physical state in response to externally applied stimuli, have found large interest as sophisticated materials that provide a complex behavior to hydrogel-based sensing devices. PMID:24957962

Computational design of a pH-sensitive IgG binding protein.

PubMed

Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David

2014-01-14

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.

Calculating the sensitivity and robustness of binding free energy calculations to force field parameters

PubMed Central

Rocklin, Gabriel J.; Mobley, David L.; Dill, Ken A.

2013-01-01

Binding free energy calculations offer a thermodynamically rigorous method to compute protein-ligand binding, and they depend on empirical force fields with hundreds of parameters. We examined the sensitivity of computed binding free energies to the ligand’s electrostatic and van der Waals parameters. Dielectric screening and cancellation of effects between ligand-protein and ligand-solvent interactions reduce the parameter sensitivity of binding affinity by 65%, compared with interaction strengths computed in the gas-phase. However, multiple changes to parameters combine additively on average, which can lead to large changes in overall affinity from many small changes to parameters. Using these results, we estimate that random, uncorrelated errors in force field nonbonded parameters must be smaller than 0.02 e per charge, 0.06 Å per radius, and 0.01 kcal/mol per well depth in order to obtain 68% (one standard deviation) confidence that a computed affinity for a moderately-sized lead compound will fall within 1 kcal/mol of the true affinity, if these are the only sources of error considered. PMID:24015114

Determination of DNA Binding Behavior of FoxA1 Constructs Using a Gold Nanoparticle-Based High Throughput Assay

NASA Astrophysics Data System (ADS)

Aung, Khin Moh Moh; Lim, Michelle Gek Liang; Hong, Shuzhen; Cheung, Edwin; Su, Xiaodi

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged-helix transcription factors. It plays crucial roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. In this study, in order to determine the regions of FoxA1 necessary for efficient Deoxyribonucleic Acid (DNA) binding, we cloned, expressed and purified a series of FoxA1 constructs that contain either the DNA Binding Domain (DBD), the Transcription Activation Domain (TAD), or both. We determined the DNA binding behavior of these constructs using traditional electrophoretic mobility shift assay (EMSA) and a recently developed gold nanoparticles (AuNPs)-based fast screening method. We conclude that just the DBD region alone is not sufficient for protein-DNA binding activity. Amino acids flanking the upstream of the DBD region are required for maximal DNA binding activity. Through this study, we have also further validated the AuNPs assay for its generality and expanded the existing protocol for comparing the DNA binding behavior of multiple proteins of different charge properties and molecular weights.

Effects of serotonin-2A receptor binding and gender on personality traits and suicidal behavior in borderline personality disorder.

PubMed

Soloff, Paul H; Chiappetta, Laurel; Mason, Neale Scott; Becker, Carl; Price, Julie C

2014-06-30

Impulsivity and aggressiveness are personality traits associated with a vulnerability to suicidal behavior. Behavioral expression of these traits differs by gender and has been related to central serotonergic function. We assessed the relationships between serotonin-2A receptor function, gender, and personality traits in borderline personality disorder (BPD), a disorder characterized by impulsive-aggression and recurrent suicidal behavior. Participants, who included 33 BPD patients and 27 healthy controls (HC), were assessed for Axis I and II disorders with the Structured Clinical Interview for DSM-IV and the International Personality Disorders Examination, and with the Diagnostic Interview for Borderline Patients-Revised for BPD. Depressed mood, impulsivity, aggression, and temperament were assessed with standardized measures. Positron emission tomography with [(18)F]altanserin as ligand and arterial blood sampling was used to determine the binding potentials (BPND) of serotonin-2A receptors in 11 regions of interest. Data were analyzed using Logan graphical analysis, controlling for age and non-specific binding. Among BPD subjects, aggression, Cluster B co-morbidity, antisocial PD, and childhood abuse were each related to altanserin binding. BPND values predicted impulsivity and aggression in BPD females (but not BPD males), and in HC males (but not HC females.) Altanserin binding was greater in BPD females than males in every contrast, but it did not discriminate suicide attempters from non-attempters. Region-specific differences in serotonin-2A receptor binding related to diagnosis and gender predicted clinical expression of aggression and impulsivity. Vulnerability to suicidal behavior in BPD may be related to serotonin-2A binding through expression of personality risk factors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

GABAergic control of neostriatal dopamine D2 receptor binding and behaviors in the rat.

PubMed

Nikolaus, Susanne; Beu, Markus; de Souza Silva, Maria Angelica; Huston, Joseph P; Antke, Christina; Müller, Hans-Wilhelm; Hautzel, Hubertus

2017-02-01

The present study assessed the influence of the GABA A receptor agonist muscimol and the GABA A receptor antagonist bicuculline on neostriatal dopamine D 2 receptor binding in relation to motor and exploratory behaviors in the rat. D 2 receptor binding was measured in baseline and after challenge with either 1mg/kg muscimol or 1mg/kg bicuculline. In additional rats, D 2 receptor binding was measured after injection of saline. After treatment with muscimol, bicuculline and saline, motor and exploratory behaviors were assessed for 30min in an open field prior to administration of [ 123 I]S-3-iodo-N-(1-ethyl-2-pyrrolidinyl)methyl-2-hydroxy-6-methoxybenzamide ([ 123 I]IBZM). For baseline and challenges, striatal equilibrium ratios (V 3 ″) were computed as estimation of the binding potential. Muscimol but not bicuculline reduced D 2 receptor binding relative to baseline and to saline. Travelled distance, duration of rearing and frequency of rearing and of head-shoulder motility were lower after muscimol compared to saline. In contrast, duration of rearing and grooming and frequency of rearing, head-shoulder motility and grooming were elevated after bicuculline relative to saline. Moreover, bicuculline decreased duration of sitting and head-shoulder motility. The muscimol-induced decrease of motor/exploratory behaviors can be related to an elevation of striatal dopamine levels. In contrast, bicuculline is likely to elicit a decline of synaptic dopamine, which, however, is compensated by the time of D 2 receptor imaging studies. The results indicate direct GABAergic control over D 2 receptor binding in the neostriatum in relation to behavioral action, and, thus, complement earlier pharmacological studies. Copyright © 2016. Published by Elsevier Inc.

Developmental Effects of Acute, Chronic, and Withdrawal from Chronic Nicotine on Fear Conditioning

PubMed Central

Portugal, George S.; Wilkinson, Derek S.; Turner, Jill R.; Blendy, Julie A.; Gould, Thomas J.

2012-01-01

Pre-adolescence and adolescence are developmental periods associated with increased vulnerability for tobacco addiction, and exposure to tobacco during these periods may lead to long-lasting changes in behavioral and neuronal plasticity. The present study examined the short- and long-term effects of nicotine and nicotine withdrawal on fear conditioning in pre-adolescent, adolescent, and adult mice, and potential underlying substrates that may mediate the developmental effects of nicotine, such as changes in nicotinic acetylcholine receptor (nAChR) binding, CREB expression, and nicotine metabolism. Age-related differences existed in sensitivity to the effects of acute nicotine, chronic nicotine and nicotine withdrawal on contextual fear conditioning (no changes in cued fear conditioning were seen); younger mice were more sensitive to the acute effects and less sensitive to the effects of nicotine withdrawal 24 hours post treatment cessation. Developmental differences in nAChR binding were associated with the effects of nicotine withdrawal on contextual learning. Developmental differences in nicotine metabolism and CREB expression were also observed, but were not related to the effects of nicotine withdrawal on contextual learning 24 hours post treatment. Chronic nicotine exposure during pre-adolescence or adolescence, however, produced long-lasting impairments in contextual learning that were observed during adulthood, whereas adult chronic nicotine exposure did not. These developmental effects could be related to changes in CREB. Overall, there is a developmental shift in the effects of nicotine on hippocampus-dependent learning and developmental exposure to nicotine results in adult cognitive deficits; these changes in cognition may play an important role in the development and maintenance of nicotine addiction. PMID:22521799

Force-extension behavior of DNA in the presence of DNA-bending nucleoid associated proteins

NASA Astrophysics Data System (ADS)

Dahlke, K.; Sing, C. E.

2018-02-01

Interactions between nucleoid associated proteins (NAPs) and DNA affect DNA polymer conformation, leading to phenomena such as concentration dependent force-extension behavior. These effects, in turn, also impact the local binding behavior of the protein, such as high forces causing proteins to unbind, or proteins binding favorably to locally bent DNA. We develop a coarse-grained NAP-DNA simulation model that incorporates both force- and concentration-dependent behaviors, in order to study the interplay between NAP binding and DNA conformation. This model system includes multi-state protein binding and unbinding, motivated by prior work, but is now dependent on the local structure of the DNA, which is related to external forces acting on the DNA strand. We observe the expected qualitative binding behavior, where more proteins are bound at lower forces than at higher forces. Our model also includes NAP-induced DNA bending, which affects DNA elasticity. We see semi-quantitative matching of our simulated force-extension behavior to the reported experimental data. By using a coarse-grained simulation, we are also able to look at non-equilibrium behaviors, such as dynamic extension of a DNA strand. We stretch a DNA strand at different rates and at different NAP concentrations to observe how the time scales of the system (such as pulling time and unbinding time) work in concert. When these time scales are similar, we observe measurable rate-dependent changes in the system, which include the number of proteins bound and the force required to extend the DNA molecule. This suggests that the relative time scales of different dynamic processes play an important role in the behavior of NAP-DNA systems.

Serotonergic and dopaminergic distinctions in the behavioral pharmacology of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD).

PubMed

Schindler, Emmanuelle A D; Dave, Kuldip D; Smolock, Elaine M; Aloyo, Vincent J; Harvey, John A

2012-03-01

After decades of social stigma, hallucinogens have reappeared in the clinical literature demonstrating unique benefits in medicine. The precise behavioral pharmacology of these compounds remains unclear, however. Two commonly studied hallucinogens, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and lysergic acid diethylamide (LSD), were investigated both in vivo and in vitro to determine the pharmacology of their behavioral effects in an animal model. Rabbits were administered DOI or LSD and observed for head bob behavior after chronic drug treatment or after pretreatment with antagonist ligands. The receptor binding characteristics of DOI and LSD were studied in vitro in frontocortical homogenates from naïve rabbits or ex vivo in animals receiving an acute drug injection. Both DOI- and LSD-elicited head bobs required serotonin(2A) (5-HT(2A)) and dopamine(1) (D(1)) receptor activation. Serotonin(2B/2C) receptors were not implicated in these behaviors. In vitro studies demonstrated that LSD and the 5-HT(2A/2C) receptor antagonist, ritanserin, bound frontocortical 5-HT(2A) receptors in a pseudo-irreversible manner. In contrast, DOI and the 5-HT(2A/2C) receptor antagonist, ketanserin, bound reversibly. These binding properties were reflected in ex vivo binding studies. The two hallucinogens also differed in that LSD showed modest D(1) receptor binding affinity whereas DOI had negligible binding affinity at this receptor. Although DOI and LSD differed in their receptor binding properties, activation of 5-HT(2A) and D(1) receptors was a common mechanism for eliciting head bob behavior. These findings implicate these two receptors in the mechanism of action of hallucinogens. Copyright © 2011 Elsevier Inc. All rights reserved.

Parallel Force Assay for Protein-Protein Interactions

PubMed Central

Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

Parallel force assay for protein-protein interactions.

PubMed

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

On the Binding Stress-Enhanced Sensitivity of (Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO3) 0.35 (PMN-PT) Piezoelectric Plate Sensor (PEPS)

NASA Astrophysics Data System (ADS)

Wu, Wei

(Pb(Mg1/3Nb2/3)O3)0.65-(PbTiO 3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) showed enhanced sensitivity in chemical and biological sensing applications which has been attributed to binding-induced crystalline orientation switching in the PMN-PT layer. However, so far there has been no direct demonstration of PEPS crystalline orientation switching upon target-analyte binding. Using biotin and streptavidin binding as a model detection system and by direct X-Ray diffraction observations after analyte binding we have unambiguously demonstrated that switching of the crystalline orientations of the PMN-PT layer indeed occurred. In addition, we have shown that PEPS sensitivity enhancement increased with an increasing transverse electromechanical coupling constant, -k31, of the PMN-PT layer--which is known to correlate with the crystalline orientation switching capability--by increasing the grain size of the PMN-PT layer or by applying a DC bias electric field. Finally, unprecedented high sensitivity of PEPS with high -k31, (i.e., -k31 > 0.3) were illustrated by the aM (10-18 M) sensitivity of in situ DNA hybridization detection without amplification and by the 100 fg/ml (10-13 g/ml) sensitivity of rapid, in situ protein detection in biological fluids such as troponin I detection in serum for early sign of myocardial infarction (heart attack), Her2 detection in serum for cancer treatment and monitoring, Tn antigen and anti-Tn antibody detection in serum for early cancer detection, and Toxins detection in stool for Clostridium difficile infection detection.

A role for calmodulin-stimulated adenylyl cyclases in cocaine sensitization.

PubMed

DiRocco, Derek P; Scheiner, Zachary S; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R

2009-02-25

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1(-/-) and AC8(-/-)) exhibit Ca(2+)-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca(2+)-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs.

A Role for Calmodulin-Stimulated Adenylyl Cyclases in Cocaine Sensitization

PubMed Central

DiRocco, Derek P.; Scheiner, Zachary S.; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R.

2009-01-01

Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that while AC1 and AC8 single knockout mice (AC1−/− and AC8−/−) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knockout (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization following chronic cocaine treatment. Because of the known role for the ERK/MAP kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated extracellular signal-regulated kinase (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase positive (ChAT+) interneurons in DKO mice relative to wild-type (WT) controls. Following acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581, and cAMP response element-binding protein (pCREB) at Ser133 following acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. PMID:19244515

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

PubMed

Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

2007-05-01

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

PubMed Central

Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

"In situ" observation of the role of chloride ion binding to monkey green sensitive visual pigment by ATR-FTIR spectroscopy.

PubMed

Katayama, Kota; Furutani, Yuji; Iwaki, Masayo; Fukuda, Tetsuya; Imai, Hiroo; Kandori, Hideki

2018-01-31

Long-wavelength-sensitive (LWS) pigment possesses a chloride binding site in its protein moiety. The binding of chloride alters the absorption spectra of LWS; this is known as the chloride effect. Although the two amino acid substitutions of His197 and Lys200 influence the chloride effect, the molecular mechanism of chloride binding, which underlies the spectral tuning, has yet to be clarified. In this study, we applied ATR-FTIR spectroscopy to monkey green (MG) pigment to gain structural information of the chloride binding site. The results suggest that chloride binding stabilizes the β-sheet structure on the extracellular side loop with perturbation of the retinal polyene chain, promotes a hydrogen bonding exchange with the hydroxyl group of Tyr, and alters the protonation state of carboxylate. Combining with the results of the binding analyses of various anions (Br - , I - and NO 3 - ), our findings suggest that the anion binding pocket is organized for only Cl - (or Br - ) to stabilize conformation around the retinal chromophore, which is functionally relevant with absorbing long wavelength light.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

PubMed

Briggman, Kevin L; Kristan, William B; González, Jesús E; Kleinfeld, David; Tsien, Roger Y

2015-01-01

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

Murine and human CFTR exhibit different sensitivities to CFTR potentiators

PubMed Central

Cui, Guiying

2015-01-01

Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as “Kalydeco.” Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by ∼13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators. PMID:26209275

An Extended Structure–Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors

PubMed Central

Feng, Wei; Zheng, Jing; Dong, Yao; Li, Xueshu; Lehmler, Hans-Joachim; Pessah, Isaac N.

2017-01-01

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure–activity relationship and a quantitative structure–activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs. PMID:27655348

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors.

PubMed

Simon, Anna J; Vallée-Bélisle, Alexis; Ricci, Francesco; Plaxco, Kevin W

2014-10-21

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.

Resistance of neuronal nitric oxide synthase-deficient mice to methamphetamine-induced dopaminergic neurotoxicity.

PubMed

Itzhak, Y; Gandia, C; Huang, P L; Ali, S F

1998-03-01

Methamphetamine (METH) is a powerful psychostimulant that produces dopaminergic neurotoxicity manifested by a decrease in the levels of dopamine, tyrosine hydroxylase activity and dopamine transporter (DAT) binding sites in the nigrostriatal system. We have recently reported that blockade of the neuronal nitric oxide synthase (nNOS) isoform by 7-nitroindazole provides protection against METH-induced neurotoxicity in Swiss Webster mice. The present study was undertaken to investigate the effect of a neurotoxic dose of METH on mutant mice lacking the nNOS gene [nNOS(-/-)] and wild-type controls. In addition, we sought to investigate the behavioral outcome of exposure to a neurotoxic dose of METH. Homozygote nNOS(-/-), heterozygote nNOS(+/-) and wild-type animals were administered either saline or METH (5 mg/kg x 3). Dopamine, DOPAC and HVA levels, as well as DAT binding site levels, were determined in striatal tissue derived 72 h after the last METH injection. This regimen of METH given to nNOS(-/-) mice affected neither the tissue content of dopamine and its metabolites nor the number of DAT binding sites. Although a moderate reduction in the levels of dopamine (35%) and DAT binding sites (32%) occurred in striatum of heterozygote nNOS(+/-) mice, a more profound depletion of the dopaminergic markers (up to 68%) was observed in the wild-type animals. METH-induced hyperthermia was observed in all animal strains examined except the nNOS(-/-) mice. Investigation of the animals' spontaneous locomotor activity before and after administration of the neurotoxic dose of METH (5 mg/kg x 3) revealed no differences. A low dose of METH (1.0 mg/kg) administered to naive animals (nNOS(-/-) and wild-type) resulted in a similar intensity of locomotor stimulation. However, 68 to 72 h after exposure to the high-dose METH regimen, a marked sensitized responses to a challenge METH injection was observed in the wild-type mice but not in the nNOS(-/-) mice. Taken together, these results indicate that nNOS(-/-) mice are protected against METH-induced dopaminergic neurotoxicity and locomotor sensitization. It also appears that a partial deficit of dopaminergic transmission in wild-type animals does not prevent the development of sensitization to METH, whereas a deficit in nNOS may attenuate this process.

Toward Improved Force-Field Accuracy through Sensitivity Analysis of Host-Guest Binding Thermodynamics

PubMed Central

Yin, Jian; Fenley, Andrew T.; Henriksen, Niel M.; Gilson, Michael K.

2015-01-01

Improving the capability of atomistic computer models to predict the thermodynamics of noncovalent binding is critical for successful structure-based drug design, and the accuracy of such calculations remains limited by non-optimal force field parameters. Ideally, one would incorporate protein-ligand affinity data into force field parametrization, but this would be inefficient and costly. We now demonstrate that sensitivity analysis can be used to efficiently tune Lennard-Jones parameters of aqueous host-guest systems for increasingly accurate calculations of binding enthalpy. These results highlight the promise of a comprehensive use of calorimetric host-guest binding data, along with existing validation data sets, to improve force field parameters for the simulation of noncovalent binding, with the ultimate goal of making protein-ligand modeling more accurate and hence speeding drug discovery. PMID:26181208

Parietal-Occipital Interactions Underlying Control- and Representation-Related Processes in Working Memory for Nonspatial Visual Features.

PubMed

Gosseries, Olivia; Yu, Qing; LaRocque, Joshua J; Starrett, Michael J; Rose, Nathan S; Cowan, Nelson; Postle, Bradley R

2018-05-02

Although the manipulation of load is popular in visual working memory research, many studies confound general attentional demands with context binding by drawing memoranda from the same stimulus category. In this fMRI study of human observers (both sexes), we created high- versus low-binding conditions, while holding load constant, by comparing trials requiring memory for the direction of motion of one random dot kinematogram (RDK; 1M trials) versus for three RDKs (3M), or versus one RDK and two color patches (1M2C). Memory precision was highest for 1M trials and comparable for 3M and 1M2C trials. And although delay-period activity in occipital cortex did not differ between the three conditions, returning to baseline for all three, multivariate pattern analysis decoding of a remembered RDK from occipital cortex was also highest for 1M trials and comparable for 3M and 1M2C trials. Delay-period activity in intraparietal sulcus (IPS), although elevated for all three conditions, displayed more sensitivity to demands on context binding than to load per se. The 1M-to-3M increase in IPS signal predicted the 1M-to-3M declines in both behavioral and neural estimates of working memory precision. These effects strengthened along a caudal-to-rostral gradient, from IPS0 to IPS5. Context binding-independent load sensitivity was observed when analyses were lateralized and extended into PFC, with trend-level effects evident in left IPS and strong effects in left lateral PFC. These findings illustrate how visual working memory capacity limitations arise from multiple factors that each recruit dissociable brain systems. SIGNIFICANCE STATEMENT Visual working memory capacity predicts performance on a wide array of cognitive and real-world outcomes. At least two theoretically distinct factors are proposed to influence visual working memory capacity limitations: an amodal attentional resource that must be shared across remembered items; and the demands on context binding. We unconfounded these two factors by varying load with items drawn from the same stimulus category ("high demands on context binding") versus items drawn from different stimulus categories ("low demands on context binding"). The results provide evidence for the dissociability, and the neural bases, of these two theorized factors, and they specify that the functions of intraparietal sulcus may relate more strongly to the control of representations than to the general allocation of attention. Copyright © 2018 the authors 0270-6474/18/384357-10$15.00/0.

A sensitive capacitive immunosensor for direct detection of human heart fatty acid-binding protein (h-FABP).

PubMed

Mihailescu, Carmen-Marinela; Stan, Dana; Iosub, Rodica; Moldovan, Carmen; Savin, Mihaela

2015-01-01

The fabrication of a capacitive interdigitated immunosensor (CID) based on a mixed self-assembled monolayer (mSAM) film for the direct detection of heart fatty-acid binding protein (h-FABP) without any labeling is described. The capacitance changes of mSAMs vs. homogenous ordered self-assembled monolayers (hSAMs) on gold work electrodes/covalently bonded antibodies/buffered medium are utilized for monitoring the specific antibody-antigen interaction. Capacitance measurements in the absence and presence of Faradaic currents were performed. The electrochemical properties of mixed monolayers were compared with those of a pure monolayer of 11-mercaptoundecanoic acid (MUA) self-assembled on gold surfaces. Taking into account the stability of the studied monolayers during the electrochemical experiments with the Faradaic process, the best SAM functionalization method was used for developing a sensitive capacitive immunosensor with a non-Faradaic process for direct immune detection of human h-FABP. Under the optimized conditions, the proposed mixed self-assembled monolayer (mSAM1) on gold electrode exhibited good insulating properties such as a capacitive behavior when detecting h-FABP from human serum in the range of 98 pg ml(-1)-100 ng ml(-1), with a detection limit of 0.836 ng ml(-1) comparative with a homogenous self-assembled monolayer (hSAM). Copyright © 2014 Elsevier B.V. All rights reserved.

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice.

PubMed

Ishige, K; Endo, H; Saito, H; Ito, Y

2001-01-19

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

An ultrasensitive micropillar-based quartz crystal microbalance device for real-time measurement of protein immobilization and protein-protein interaction.

PubMed

Su, Junwei; Esmaeilzadeh, Hamed; Zhang, Fang; Yu, Qing; Cernigliaro, George; Xu, Jin; Sun, Hongwei

2018-01-15

A new sensing device was developed to achieve ultrahigh sensitivity, by coupling polymer micropillars with a quartz crystal microbalance (QCM) substrate to form a two-degree- of-freedom resonance system (QCM-P). The sensitivity of these QCM-P devices was evaluated by measuring mass changes for both deposited gold film and adsorption of bovine serum albumin (BSA), respectively, on poly(methyl methacrylate) (PMMA) micropillar surfaces, as well as assessing ligand-analyte binding interactions between anti-human immunoglobulin G (anti-hIgG) and human immunoglobulin G (hIgG). The anti-hIgG and hIgG binding results show QCM-P achieved an eightfold improvement in sensitivity relative to conventional QCM sensors. In addition, the binding affinity obtained from the QCM-P device for anti-hIgG and hIgG proteins was found in good agreement with that measured by surface plasmon resonance (SPR) for the same binding reaction. Copyright © 2017 Elsevier B.V. All rights reserved.

Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior.

PubMed

Hockenberry, Alyson M; Hutchens, Danielle M; Agellon, Al; So, Magdalene

2016-12-06

Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. ΔpilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilT L201C ). Purified PilT L201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilT L201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilT L201C cells is intermediate between the behaviors of wt and ΔpilT cells. The infection behavior of pilT L201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se, is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme. Copyright © 2016 Hockenberry et al.

Neutron star radii, universal relations, and the role of prior distributions

DOE PAGES

Steiner, Andrew W.; Lattimer, James M.; Brown, Edward F.

2016-02-02

We investigate constraints on neutron star structure arising from the assumptions that neutron stars have crusts, that recent calculations of pure neutron matter limit the equation of state of neutron star matter near the nuclear saturation density, that the high-density equation of state is limited by causality and the largest high-accuracy neutron star mass measurement, and that general relativity is the correct theory of gravity. We explore the role of prior assumptions by considering two classes of equation of state models. In a first, the intermediate- and high-density behavior of the equation of state is parameterized by piecewise polytropes. Inmore » the second class, the high-density behavior of the equation of state is parameterized by piecewise continuous line segments. The smallest density at which high-density matter appears is varied in order to allow for strong phase transitions above the nuclear saturation density. We critically examine correlations among the pressure of matter, radii, maximum masses, the binding energy, the moment of inertia, and the tidal deformability, paying special attention to the sensitivity of these correlations to prior assumptions about the equation of state. It is possible to constrain the radii of 1.4 solar mass neutron stars to be larger than 10 km, even without consideration of additional astrophysical observations, for example, those from photospheric radius expansion bursts or quiescent low-mass X-ray binaries. We are able to improve the accuracy of known correlations between the moment of inertia and compactness as well as the binding energy and compactness. Furthermore, we also demonstrate the existence of a correlation between the neutron star binding energy and the moment of inertia.« less

Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats.

PubMed

Melas, Philippe A; Qvist, Johanna S; Deidda, Matteo; Upreti, Chirag; Wei, Ya Bin; Sanna, Fabrizio; Fratta, Walter; Scherma, Maria; Fadda, Paola; Kandel, Denise B; Kandel, Eric R

2018-03-13

Reduced eukaryotic Initiation Factor 2 (eIF2)α phosphorylation (p-eIF2α) enhances protein synthesis, memory formation, and addiction-like behaviors. However, p-eIF2α has not been examined with regard to psychoactive cannabinoids and cross-sensitization. Here, we find that a cannabinoid receptor agonist (WIN 55,212-2 mesylate [WIN]) reduced p-eIF2α in vitro by upregulating GADD34 (PPP1R15A), the recruiter of protein phosphatase 1 (PP1). The induction of GADD34 was linked to ERK/CREB signaling and to CREB-binding protein (CBP)-mediated histone hyperacetylation at the Gadd34 locus. In vitro, WIN also upregulated eIF2B1, an eIF2 activator subunit. We next found that WIN administration in vivo reduced p-eIF2α in the nucleus accumbens of adolescent, but not adult, rats. By contrast, WIN increased dorsal striatal levels of eIF2B1 and ΔFosB among both adolescents and adults. In addition, we found cross-sensitization between WIN and cocaine only among adolescents. These findings show that cannabinoids can modulate eukaryotic initiation factors, and they suggest a possible link between p-eIF2α and the gateway drug properties of psychoactive cannabinoids. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Maternal BIS Sensitivity, Overprotective Parenting, and Children’s Internalizing Behaviors

PubMed Central

Kiel, Elizabeth J.; Maack, Danielle J.

2012-01-01

Although sensitivity to the Behavioral Inhibition System within Gray’s (1970) reinforcement sensitivity theory relates to individuals’ own depressive and anxious symptomatology, less is known about how parental BIS sensitivity relates to early indicators of internalizing problems in young children. Moreover, the extent to which this parental characteristic relates to parenting behavior, and children’s internalizing problems above and beyond parenting, remains unknown. The current study assessed maternal BIS sensitivity, overprotective parenting, and toddlers’ internalizing behaviors in a sample of 91 mothers while controlling for mothers’ own internalizing symptomatology. Heightened BIS sensitivity related to both overprotective parenting and internalizing behaviors. Overprotective parenting partially mediated the relation between BIS sensitivity and children’s internalizing behaviors, although BIS sensitivity maintained a marginal relation to internalizing behaviors. Maternal BIS sensitivity and toddler internalizing behaviors may represent a shared disposition towards inhibition that is somewhat accounted for by overprotective parenting. PMID:22904590

Evidence for sugar addiction: Behavioral and neurochemical effects of intermittent, excessive sugar intake

PubMed Central

Avena, Nicole M.; Rada, Pedro; Hoebel, Bartley G.

2008-01-01

The experimental question is whether or not sugar can be a substance of abuse and lead to a natural form of addiction. “Food addiction” seems plausible because brain pathways that evolved to respond to natural rewards are also activated by addictive drugs. Sugar is noteworthy as a substance that releases opioids and dopamine and thus might be expected to have addictive potential. This review summarizes evidence of sugar dependence in an animal model. Four components of addiction are analyzed. “Bingeing”, “withdrawal”, “craving” and cross-sensitization are each given operational definitions and demonstrated behaviorally with sugar bingeing as the reinforcer. These behaviors are then related to neurochemical changes in the brain that also occur with addictive drugs. Neural adaptations include changes in dopamine and opioid receptor binding, enkephalin mRNA expression and dopamine and acetylcholine release in the nucleus accumbens. The evidence supports the hypothesis that under certain circumstances rats can become sugar dependent. This may translate to some human conditions as suggested by the literature on eating disorders and obesity. PMID:17617461

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus.

PubMed

Hocking, Jason; Priyadarshini, Richa; Takacs, Constantin N; Costa, Teresa; Dye, Natalie A; Shapiro, Lucy; Vollmer, Waldemar; Jacobs-Wagner, Christine

2012-06-01

The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.

A study of planar anchor groups for graphene-based single-molecule electronics.

PubMed

Bailey, Steven; Visontai, David; Lambert, Colin J; Bryce, Martin R; Frampton, Harry; Chappell, David

2014-02-07

To identify families of stable planar anchor groups for use in single molecule electronics, we report detailed results for the binding energies of two families of anthracene and pyrene derivatives adsorbed onto graphene. We find that all the selected derivatives functionalized with either electron donating or electron accepting substituents bind more strongly to graphene than the parent non-functionalized anthracene or pyrene. The binding energy is sensitive to the detailed atomic alignment of substituent groups over the graphene substrate leading to larger than expected binding energies for -OH and -CN derivatives. Furthermore, the ordering of the binding energies within the anthracene and pyrene series does not simply follow the electron affinities of the substituents. Energy barriers to rotation or displacement on the graphene surface are much lower than binding energies for adsorption and therefore at room temperature, although the molecules are bound to the graphene, they are almost free to move along the graphene surface. Binding energies can be increased by incorporating electrically inert side chains and are sensitive to the conformation of such chains.

A study of planar anchor groups for graphene-based single-molecule electronics

NASA Astrophysics Data System (ADS)

Bailey, Steven; Visontai, David; Lambert, Colin J.; Bryce, Martin R.; Frampton, Harry; Chappell, David

2014-02-01

To identify families of stable planar anchor groups for use in single molecule electronics, we report detailed results for the binding energies of two families of anthracene and pyrene derivatives adsorbed onto graphene. We find that all the selected derivatives functionalized with either electron donating or electron accepting substituents bind more strongly to graphene than the parent non-functionalized anthracene or pyrene. The binding energy is sensitive to the detailed atomic alignment of substituent groups over the graphene substrate leading to larger than expected binding energies for -OH and -CN derivatives. Furthermore, the ordering of the binding energies within the anthracene and pyrene series does not simply follow the electron affinities of the substituents. Energy barriers to rotation or displacement on the graphene surface are much lower than binding energies for adsorption and therefore at room temperature, although the molecules are bound to the graphene, they are almost free to move along the graphene surface. Binding energies can be increased by incorporating electrically inert side chains and are sensitive to the conformation of such chains.

An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy

PubMed Central

Selb, R.; Eckl-Dorna, J.; Vrtala, S.; Valenta, R.; Niederberger, V.

2017-01-01

Background It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy. Methods In 18 patients treated by Bet v 1-fragment-specific immunotherapy, the effects of IgG antibodies specific for the fragments on the binding of IgE antibodies to Bet v 1 were measured by ELISA. Blocking and possible enhancing effects on IgE binding were compared with skin sensitivity to Bet v 1 after treatment. Results We found that fragment-specific IgG enhanced IgE binding to Bet v 1 in two patients who also showed an increase of skin sensitivity to Bet v 1. Conclusion Our results indicate that it may be possible to develop serological tests which predict the induction of unfavourable IgG antibodies enhancing the binding of IgE to Bet v 1 during immunotherapy. PMID:23998344

Highly variable sensitivity of five binding and two bio-assays for TSH-receptor antibodies.

PubMed

Diana, T; Wüster, C; Kanitz, M; Kahaly, G J

2016-10-01

TSH-receptor (TSHR) antibodies (Ab) can be measured with binding or bio-assays. Sensitivity and specificity of five binding and two bio-assays were compared. TSHR-blocking (TBAb) and TSHR-stimulating (TSAb) Ab were measured with reporter bio-assays. Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bTSH alone. TSAb was reported as percentage of specimen-to-reference ratio (SRR%). TSHR-binding inhibitory immunoglobulins (TBII) were measured with Kronus, Dynex, Kryptor, Cobas, and Immulite. Sixty patients with Graves' disease (GD), 20 with Hashimoto's thyroiditis (HT), and 20 healthy controls (C) were included. C tested negative in all assays (specificity 100 %) while all 60 hyperthyroid GD patients tested positive in the TSAb bio-assay (sensitivity 100 %). Among these 60 GD patients, 20 had low TSAb positivity (SRR% 140-279), but were TBII positive in only 20 (100 %), 7 (35 %), 9 (45 %), 11 (55 %), and 18 (90 %) using the Kronus, Dynex, Kryptor, Cobas, and Immulite, respectively. In 20 moderate TSAb-positive (SRR% 280-420) patients, TBII tested positive in 20 (100 %), 14 (70 %), 13 (65 %), 16 (80 %), and 19 (95 %), respectively. The high (SRR% > 420) TSAb-positive patients were all TBII positive. All 20 hypothyroid HT patients tested TBAb positive (sensitivity 100 %) in the bio-assay while they tested TBII positive in 20 (100 %), 18 (90 %), 20, 20, and 18, respectively. Results obtained with two luminometers correlated for TSAb positive (r = 0.99, p < 0.001), TBAb positive (r = 0.88, p < 0.001), and C (r = 0.86, p < 0.001). None of the binding assays differentiated between TSAb and TBAb. Sensitivity is highly variable between binding and bio-assays for TSHR-Abs.

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

PubMed

Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

2014-01-01

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.

Detailed Analysis of the Binding Mode of Vanilloids to Transient Receptor Potential Vanilloid Type I (TRPV1) by a Mutational and Computational Study

PubMed Central

Mori, Yoshikazu; Ogawa, Kazuo; Warabi, Eiji; Yamamoto, Masahiro; Hirokawa, Takatsugu

2016-01-01

Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and a multimodal sensor protein. Since the precise structure of TRPV1 was obtained by electron cryo-microscopy, the binding mode of representative agonists such as capsaicin and resiniferatoxin (RTX) has been extensively characterized; however, detailed information on the binding mode of other vanilloids remains lacking. In this study, mutational analysis of human TRPV1 was performed, and four agonists (capsaicin, RTX, [6]-shogaol and [6]-gingerol) were used to identify amino acid residues involved in ligand binding and/or modulation of proton sensitivity. The detailed binding mode of each ligand was then simulated by computational analysis. As a result, three amino acids (L518, F591 and L670) were newly identified as being involved in ligand binding and/or modulation of proton sensitivity. In addition, in silico docking simulation and a subsequent mutational study suggested that [6]-gingerol might bind to and activate TRPV1 in a unique manner. These results provide novel insights into the binding mode of various vanilloids to the channel and will be helpful in developing a TRPV1 modulator. PMID:27606946

Behavioral studies with anxiolytic drugs. IV. Serotonergic involvement in the effects of buspirone on punished behavior of pigeons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witkin, J.M.; Mansbach, R.S.; Barrett, J.E.

1987-12-01

Interactions of the nonbenzodiazepine anxiolytic, buspirone, with serotonin (5-HT) were studied using behavioral and neurochemical procedures. Punished responding was studied in pigeons as this behavior is a generally acknowledged preclinical predictor of anxiolytic activity and because buspirone increases punished responding of pigeons with greater potency and efficacy than in other species. Keypeck responses were maintained under either fixed-interval or fixed-ratio schedules of food presentation; every 30th response produced a brief electric shock and suppressed responding (punishment). Buspirone (0.1-5.6 mg/kg i.m.) produced dose-related increases in punished responding which reached a maximum at 1 mg/kg. A serotonin agonist, MK-212 (0.01 mg/kg), antagonizedmore » whereas the 5-HT antagonist, cyproheptadine (0.01 mg/kg), potentiated the effects of buspirone without having behavioral effects of their own. The characteristics of (/sup 3/H)-5-HT binding in pigeon brain membranes were similar to results reported in mammalian brain. Neither buspirone, MJ-13805 (gepirone, a related analog), nor MJ-13653 (a buspirone metabolite), significantly affected (/sup 3/H)-5-HT binding and none of the compounds appreciably inhibited uptake of (/sup 3/H)-5-HT into pigeon cerebral synaptosomes. Hill coefficients significantly less than unity for all drugs except 5-HT suggested multiple serotonergic binding sites for buspirone and analogs. Buspirone and MJ-13805 (1 nM) inhibited (/sup 3/H)ketanserin binding (a measure of 5-HT2 binding sites) in pigeon cerebrum with Ki values above 10(-6) M. The number of (/sup 3/H)ketanserin binding sites was estimated to be 109 fmol/mg of protein in pigeon cerebrum compared to 400 fmol/mg of protein in rat cerebrum.« less

A novel and sensitive radioreceptor assay for serum melatonin levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tenn, C.; Niles, L.

A simple and sensitive radioreceptor assay (RRA) has been developed to measure melatonin levels in serum. The assay is based on competition between 2-({sup 125}I)iodomelatonin (({sup 125}I)MEL) and melatonin for binding to high-affinity binding sites in chick forebrain. To measure the amount of melatonin present in a serum sample, it was extracted with dichloromethane and added to the assay medium. The percentage inhibition of radioligand binding in the presence of the extracted serum was determined and compared to the percent displacement by known amounts of melatonin in a standard curve. There was little or no cross-reactivity with other structurally relatedmore » compounds. The sensitivity of the assay is {approximately}1.5pg/0.15 mL and the intra- and inter-assay variations are approximately 8%. Since the RRA results are comparable to that of an established radioimmunoassay (RIA), it provides a sensitive and rapid alternative to the more time consuming RIA.« less

Plant cell pH-static circuit mediated by fusicoccin-binding proteins.

PubMed

Drabkin, A V; Trofimova, M S; Smolenskaya, I N; Klychnikov, O I; Chelysheva, V V; Babakov, A V

1997-03-24

On sugar beet protoplasts that carry two types of fusicoccin-binding sites, a pH downshift in a physiological range (7.0-6.6) markedly enhanced the efficiency of fusicoccin (FC) binding, mainly owing to increased avidity of low-affinity FC-binding sites. This may allow the FC-binding proteins to act as pH-sensitive modulators of cell activity, for instance, via plasma membrane H+-ATPase or potassium channels.

Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

PubMed

van der Vaart, Arjan

2015-05-01

Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in most performance measures. To the best of our knowledge, this is the first sequence-based prediction of protein-binding nucleotides in RNA which considers the binding partner of RNA. The new model will provide valuable information for designing biochemical experiments to find putative protein-binding sites in RNA with unknown structure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In vitro sensitivity of cholinesterases and [3H]oxotremorine-M binding in heart and brain of adult and aging rats to organophosphorus anticholinesterases.

PubMed

Mirajkar, Nikita; Pope, Carey N

2008-10-15

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [(3)H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [(3)H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders.

IN VITRO SENSITIVITY OF CHOLINESTERASES AND [3H]OXOTREMORINE-M BINDING IN HEART AND BRAIN OF ADULT AND AGING RATS TO ORGANOPHOSPHORUS ANTICHOLINESTERASES

PubMed Central

Mirajkar, Nikita; Pope, Carey N.

2008-01-01

Organophosphorus (OP) insecticides elicit toxicity via acetylcholinesterase inhibition, allowing acetylcholine accumulation and excessive stimulation of cholinergic receptors. Some OP insecticides bind to additional macromolecules including butyrylcholinesterase and cholinergic receptors. While neurotoxicity from OP anticholinesterases has been extensively studied, effects on cardiac function have received less attention. We compared the in vitro sensitivity of acetylcholinesterase, butyrylcholinesterase and [3H]oxotremorine-M binding to muscarinic receptors in the cortex and heart of adult (3 months) and aging (18 months) rats to chlorpyrifos, methyl parathion and their active metabolites chlorpyrifos oxon and methyl paraoxon. Using selective inhibitors, the great majority of cholinesterase in brain was defined as acetylcholinesterase, while butyrylcholinesterase was the major cholinesterase in heart, regardless of age. In the heart, butyrylcholinesterase was markedly more sensitive than acetylcholinesterase to inhibition by chlorpyrifos oxon, and butyrylcholinesterase in tissues from aging rats was more sensitive than enzyme from adults, possibly due to differences in A-esterase mediated detoxification. Relatively similar differences were noted in brain. In contrast, acetylcholinesterase was more sensitive than butyrylcholinesterase to methyl paraoxon in both heart and brain, but no age-related differences were noted. Both oxons displaced [3H]oxotremorine-M binding in heart and brain of both age groups in a concentration-dependent manner. Chlorpyrifos had no effect but methyl parathion was a potent displacer of binding in heart and brain of both age groups. Such OP and age-related differences in interactions with cholinergic macromolecules may be important because of potential for environmental exposures to insecticides as well as the use of anticholinesterases in age-related neurological disorders. PMID:18761328

Evaluation of OASIS QSAR Models Using ToxCast™ in Vitro Estrogen and Androgen Receptor Binding Data and Application in an Integrated Endocrine Screening Approach

PubMed Central

Bhhatarai, Barun; Wilson, Daniel M.; Price, Paul S.; Marty, Sue; Parks, Amanda K.; Carney, Edward

2016-01-01

Background: Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed. Objectives: We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 μM), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay. Methods: NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined. Results: The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92–100%) and specificity (70–81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 μM, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 μM were active in the uterotrophic assay. Conclusions: We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had high sensitivity and specificity when compounds were in-domain of the models. Based on this research, we recommend a tiered screening approach wherein a) QSAR is used to identify compounds in-domain of the ER or AR binding models and predicted to bind; b) those compounds are screened in vitro to assess binding potency; and c) the stronger binders (AC50 < 1 μM) are screened in vivo. This scheme prioritizes compounds for integrative testing and risk assessment. Importantly, compounds that are not in-domain, that are predicted either not to bind or to bind weakly, that are not active in in vitro, that require metabolism to manifest activity, or for which in vivo AR testing is in order, need to be assessed differently. Citation: Bhhatarai B, Wilson DM, Price PS, Marty S, Parks AK, Carney E. 2016. Evaluation of OASIS QSAR models using ToxCast™ in vitro estrogen and androgen receptor binding data and application in an integrated endocrine screening approach. Environ Health Perspect 124:1453–1461; http://dx.doi.org/10.1289/EHP184 PMID:27152837

Intranasal Dopamine Reduces In Vivo [(123)I]FP-CIT Binding to Striatal Dopamine Transporter: Correlation with Behavioral Changes and Evidence for Pavlovian Conditioned Dopamine Response.

PubMed

de Souza Silva, Maria A; Mattern, Claudia; Decheva, Cvetana; Huston, Joseph P; Sadile, Adolfo G; Beu, Markus; Müller, H-W; Nikolaus, Susanne

2016-01-01

Dopamine (DA), which does not cross the blood-brain barrier, has central and behavioral effects when administered via the nasal route. Neither the mechanisms of central action of intranasal dopamine (IN-DA), nor its mechanisms of diffusion and transport into the brain are well understood. We here examined whether IN-DA application influences dopamine transporter (DAT) binding in the dorsal striatum and assessed the extent of binding in relation to motor and exploratory behaviors. We hypothesized that, based on the finding of increased extracellular DA in the striatum induced by application of IN-DA, binding of [(123)I]FP-CIT to the DAT should be decreased due to competition at the receptor. Rats were administered 3 mg/kg IN-DA and vehicle (VEH), with IN-DA injection either preceding or following VEH. Then motor and exploratory behaviors (traveled distance, velocity, center time, sitting, rearing, head-shoulder motility, grooming) were assessed for 30 min in an open field prior to administration of [(123)I]FP-CIT. DAT binding after IN-DA and VEH was measured with small animal SPECT 2 h following administration of the radioligand. (1) After IN-DA application, striatal DAT binding was significantly lower as compared to VEH, indicating that the nasally delivered DA had central action and increased DA levels comparable to that found previously with L-DOPA administration; and (2) DAT binding in response to intranasal VEH was lower when IN-DA application preceded VEH treatment. This finding is suggestive of Pavlovian conditioning of DA at the level of the DAT, since the DA treatment modified (decreased) the binding in response to the subsequent VEH treatment. VEH treatment also reduced motor and exploratory behaviors more when applied before, as compared to when it followed IN-DA application, also indicative of behavioral Pavlovian conditioning akin to that found upon application of various psychostimulant drugs. (a) demonstrate a direct central action of intranasally applied DA on the DAT in the dorsal striatum, indicating enhanced DA availability; and (b) provide first evidence of a Pavlovian conditioned DA response at the DAT. The latter results have relevance to understanding neurochemical mechanisms that underlie placebo action in the treatment of Parkinsonian patients.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using (/sup 125/I)FK33824

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rothman, R.B.; Jacobson, A.E.; Rice, K.C.

1987-11-01

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites (25,35), consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, (/sup 3/H)oxymorphone, (/sup 3/H)D-ala2-MePhe4, Gly-ol5-enkephalin (DAGO), and (/sup 125/I)D-ala2-Me-Phe4-met(o)-ol)enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu bindingmore » sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand (/sup 125/I)FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.« less

Role of electron transfer in Ce{sup 3+} sensitized Yb{sup 3+} luminescence in borate glass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontakke, Atul D., E-mail: sontakke.atul.55a@st.kyoto-u.ac.jp; Katayama, Yumiko; Zhuang, Yixi

2015-01-07

In a Ce{sup 3+}-Yb{sup 3+} system, two mechanisms are proposed so far namely, the quantum cutting mechanism and the electron transfer mechanism explaining Yb{sup 3+} infrared luminescence under Ce{sup 3+} excitation. Among them, the quantum cutting mechanism, where one Ce{sup 3+} photon (ultraviolet/blue) gives rise to two Yb{sup 3+} photons (near infrared) is widely sought for because of its huge potential in enhancing the solar cell efficiency. In present study on Ce{sup 3+}-Yb{sup 3+} codoped borate glasses, Ce{sup 3+} sensitized Yb{sup 3+} luminescence at ∼1 μm have been observed on Ce{sup 3+} 5d state excitation. However, the intensity of sensitized Yb{supmore » 3+} luminescence is found to be very weak compared to the strong quenching occurred in Ce{sup 3+} luminescence in Yb{sup 3+} codoped glasses. Moreover, the absolute luminescence quantum yield also showed a decreasing trend with Yb{sup 3+} codoping in the glasses. The overall behavior of the luminescence properties and the quantum yield is strongly contradicting with the quantum cutting phenomenon. The results are attributed to the energetically favorable electron transfer interactions followed by Ce{sup 3+}-Yb{sup 3+} ⇌ Ce{sup 4+}-Yb{sup 2+} inter-valence charge transfer and successfully explained using the absolute electron binding energies of dopant ions in the studied borate glass. Finally, an attempt has been presented to generalize the electron transfer mechanism among opposite oxidation/reduction property dopant ions using the vacuum referred electron binding energy (VRBE) scheme for lanthanide series.« less

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity.

PubMed

Serra, Montserrat; Brazís, Pilar; Fondati, Alessandra; Puigdemont, Anna

2006-11-01

To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. 8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

Positron emission tomography quantification of serotonin transporter in suicide attempters with major depressive disorder.

PubMed

Miller, Jeffrey M; Hesselgrave, Natalie; Ogden, R Todd; Sullivan, Gregory M; Oquendo, Maria A; Mann, J John; Parsey, Ramin V

2013-08-15

Several lines of evidence implicate abnormal serotonergic function in suicidal behavior and completed suicide, including low serotonin transporter binding in postmortem studies of completed suicide. We have also reported low in vivo serotonin transporter binding in major depressive disorder (MDD) during a major depressive episode using positron emission tomography (PET) with [(11)C]McN5652. We quantified regional brain serotonin transporter binding in vivo in depressed suicide attempters, depressed nonattempters, and healthy controls using PET and a superior radiotracer, [(11)C]DASB. Fifty-one subjects with DSM-IV current MDD, 15 of whom were past suicide attempters, and 32 healthy control subjects underwent PET scanning with [(11)C]DASB to quantify in vivo regional brain serotonin transporter binding. Metabolite-corrected arterial input functions and plasma free-fraction were acquired to improve quantification. Depressed suicide attempters had lower serotonin transporter binding in midbrain compared with depressed nonattempters (p = .031) and control subjects (p = .0093). There was no difference in serotonin transporter binding comparing all depressed subjects with healthy control subjects considering six a priori regions of interest simultaneously (p = .41). Low midbrain serotonin transporter binding appears to be related to the pathophysiology of suicidal behavior rather than of major depressive disorder. This is consistent with postmortem work showing low midbrain serotonin transporter binding capacity in depressed suicides and may partially explain discrepant in vivo findings quantifying serotonin transporter in depression. Future studies should investigate midbrain serotonin transporter binding as a predictor of suicidal behavior in MDD and determine the cause of low binding. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Muscarinic binding sites in cultured bovine pulmonary arterial endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aronstam, R.S.; Catravas, J.D.; Ryan, U.S.

The authors have previously reported a) the presence of muscarinic binding sites on cultured bovine pulmonary arterial endothelial cells (BPAE; 2,000 sites/cell) and b) that acetylcholine inhibits the release of thromboxane B/sub 2/ fro BPAE. Since the authors findings could reflect muscarinic receptors (mAChR) on BPAE, they have further investigated the nature of BPAE muscarinic binding sites and contrast them to those of known functional mAChR. Muscarinic binding sites on BPAE resembled mAChR in that a) the binding of 3 nM /sup 3/H QNB was inhibited by muscarinic agonists and antagonists; b) /sup 3/H QNB binding was 30 times moremore » sensitive to R(-)- than to S(+)-QNB; c) carbamylcholine binding was resolved into high and low affinity components (IC50's = 0.04 and 2 ..mu..M; d) 5'-guanylylimidodiphosphate (100 ..mu..M) shifted agonist binding curves to the right by a factor of 3; 4) the atropine-sensitive binding of /sup 3/H oxotremorine-M (/sup 3/H-OXO-M) was depressed by the guanine nucleotide (IC50 + 60 ..mu..M). However, although gallamine allosterically regulates mAChR binding in other tissues, it did not affect the rates of dissociation of /sup 3/H QNB, /sup 3/H methylscopolamine or /sup 3/H OXO-M from BPAE binding sites. Thus, BPAE muscarinic binding sites posses many but not all of the properties associated with functional mAChR.« less

GABAB Receptor Positive Modulation Decreases Selective Molecular and Behavioral Effects of Cocaine

PubMed Central

Lhuillier, Loic; Mombereau, Cedric; Cryan, John F.; Kaupmann, Klemens

2006-01-01

Exposure to cocaine induces selective behavioral and molecular adaptations. In rodents, acute cocaine induces increased locomotor activity whereas prolonged drug exposure results in behavioral locomotor sensitization, which is thought to be a consequence of drug–induced neuroadaptive changes. Recent attention has been given to compounds activating GABAB receptors as potential anti-addictive therapies. In particular the principle of allosteric positive GABAB receptor modulators is very promising in this respect, as positive modulators lack the sedative and muscle relaxant properties of full GABAB receptor agonists such as baclofen. Here we investigated the effects of systemic application of the GABAB receptor positive modulator GS39783 in animals treated with acute and chronic cocaine administration. Both GS39783 and baclofen dose-dependently attenuated acute cocaine-induced hyperlocomotion. Furthermore, both compounds also efficiently blocked cocaine-induced Fos induction in the striatal complex. In chronic studies GS39783 induced a modest attenuation of cocaine-induced locomotor sensitization. Chronic cocaine induces the accumulation of the transcription factor ΔFosB and up regulates cAMP-response-element-binding-protein (CREB) and dopamine-and-cAMP-regulated-phosphoprotein of 32 kd (DARPP-32). GS39783 blocked the induction/activation of DARPP-32 and CREB in the nucleus accumbens and dorsal striatum and partially inhibited ΔFosB accumulation in the dorsal striatum. In summary our data provide evidence that GS39783 attenuates the acute behavioral effects of cocaine exposure in rodents and in addition prevents the induction of selective long-term adaptive changes in dopaminergic signaling pathways. Further investigation of GABAB receptor positive modulation as a novel therapeutic strategy for the treatment of cocaine dependence and possibly other drugs of abuse is therefore warranted. PMID:16710312

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

2000-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Isolation of a pH-Sensitive IgNAR Variable Domain from a Yeast-Displayed, Histidine-Doped Master Library.

PubMed

Könning, Doreen; Zielonka, Stefan; Sellmann, Carolin; Schröter, Christian; Grzeschik, Julius; Becker, Stefan; Kolmar, Harald

2016-04-01

In recent years, engineering of pH-sensitivity into antibodies as well as antibody-derived fragments has become more and more attractive for biomedical and biotechnological applications. Herein, we report the isolation of the first pH-sensitive IgNAR variable domain (vNAR), which was isolated from a yeast-displayed, semi-synthetic master library. This strategy enables the direct identification of pH-dependent binders from a histidine-enriched CDR3 library. Displayed vNAR variants contained two histidine substitutions on average at random positions in their 12-residue CDR3 loop. Upon screening of seven rounds against the proof-of-concept target EpCAM (selection for binding at pH 7.4 and decreased binding at pH 6.0), a single clone was obtained that showed specific and pH-dependent binding as characterized by yeast surface display and biolayer interferometry. Potential applications for such pH-dependent vNAR domains include their employment in tailored affinity chromatography, enabling mild elution protocols. Moreover, utilizing a master library for the isolation of pH-sensitive vNAR variants may be a generic strategy to obtain binding entities with prescribed characteristics for applications in biotechnology, diagnostics, and therapy.

Silicon nanowire field-effect transistors for the detection of proteins

NASA Astrophysics Data System (ADS)

Madler, Carsten

In this dissertation I present results on our efforts to increase the sensitivity and selectivity of silicon nanowire ion-sensitive field-effect transistors for the detection of biomarkers, as well as a novel method for wireless power transfer based on metamaterial rectennas for their potential use as implantable sensors. The sensing scheme is based on changes in the conductance of the semiconducting nanowires upon binding of charged entities to the surface, which induces a field-effect. Monitoring the differential conductance thus provides information of the selective binding of biological molecules of interest to previously covalently linked counterparts on the nanowire surface. In order to improve on the performance of the nanowire sensing, we devised and fabricated a nanowire Wheatstone bridge, which allows canceling out of signal drift due to thermal fluctuations and dynamics of fluid flow. We showed that balancing the bridge significantly improves the signal-to-noise ratio. Further, we demonstrated the sensing of novel melanoma biomarker TROY at clinically relevant concentrations and distinguished it from nonspecific binding by comparing the reaction kinetics. For increased sensitivity, an amplification method was employed using an enzyme which catalyzes a signal-generating reaction by changing the redox potential of a redox pair. In addition, we investigated the electric double layer, which forms around charges in an electrolytic solution. It causes electrostatic screening of the proteins of interest, which puts a fundamental limitation on the biomarker detection in solutions with high salt concentrations, such as blood. We solved the coupled Nernst-Planck and Poisson equations for the electrolyte under influence of an oscillating electric field and discovered oscillations of the counterion concentration at a characteristic frequency. In addition to exploring different methods for improved sensing capabilities, we studied an innovative method to supply power to implantable biosensors wirelessly, eliminating the need for batteries. A metamaterial split ring resonator is integrated with a rectifying circuit for efficient conversion of microwave radiation to direct electrical power. We studied the near-field behavior of this rectenna with respect to distance, polarization, power, and frequency. Using a 100 mW microwave power source, we demonstrated operating a simple silicon nanowire pH sensor with light indicator.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ying

My graduate research has focused on separation science and bioanalytical analysis, which emphasized in method development. It includes three major areas: enantiomeric separations using high performance liquid chromatography (HPLC), Super/subcritical fluid chromatography (SFC), and capillary electrophoresis (CE); drug-protein binding behavior studies using CE; and carbohydrate analysis using liquid chromatograph-electrospray ionization mass spectrometry (LC-ESI-MS). Enantiomeric separations continue to be extremely important in the pharmaceutical industry. An in-depth evaluation of the enantiomeric separation capabilities of macrocyclic glycopeptides CSPs with SFC mobile phases was investigated using a set of over 100 chiral compounds. It was found that the macrocyclic based CSPs were ablemore » to separate enantiomers of various compounds with different polarities and functionalities. Seventy percent of all separations were achieved in less than 4 min due to the high flow rate (4.0 ml/min) that can be used in SFC. Drug-protein binding is an important process in determining the activity and fate of a drug once it enters the body. Two drug/protein systems have been studied using frontal analysis CE method. More sensitive fluorescence detection was introduced in this assay, which overcame the problem of low sensitivity that is common when using UV detection for drug-protein studies. In addition, the first usage of an argon ion laser with 257 nm beam coupled with CCD camera as a frontal analysis detection method enabled the simultaneous observation of drug fluorescence as well as the protein fluorescence. LC-ESI-MS was used for the separation and characterization of underivatized oligosaccharide mixtures. With the limits of detection as low as 50 picograms, all individual components of oligosaccharide mixtures (up to 11 glucose-units long) were baseline resolved on a Cyclobond I 2000 column and detected using ESI-MS. This system is characterized by high chromatographic resolution, high column stability, and high sensitivity. In addition, this method showed potential usefulness for the sensitive and quick analysis of hydrolysis products of polysaccharides, and for trace level analysis of individual oligosaccharides or oligosaccharide isomers from biological systems.« less

Pacemaker-neuron–dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation

PubMed Central

Lee, Euna; Cho, Eunjoo; Kang, Doo Hyun; Jeong, Eun Hee; Chen, Zheng; Yoo, Seung-Hee; Kim, Eun Young

2016-01-01

Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657–707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box–dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657–707 deletion in the Clock (Clkout) genetic background (p{dClk-Δ};Clkout), oscillation of core clock genes’ mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657–707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clkout flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clkout flies showed pacemaker-neuron–dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clkout flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657–707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions. PMID:27489346

Using NMR Spectroscopy to Investigate the Solution Behavior of Nerve Agents and Their Binding to Acetylcholinesterase

DTIC Science & Technology

2016-01-01

USING NMR SPECTROSCOPY TO INVESTIGATE THE SOLUTION BEHAVIOR OF NERVE AGENTS AND THEIR BINDING TO...XX-01-2016 2. REPORT TYPE Final 3. DATES COVERED (From - To) Jan – Jun 2015 4. TITLE AND SUBTITLE Using NMR Spectroscopy to Investigate the...MOLECULAR MOTIONS AND NMR SPECTROSCOPY ...................................................................................................3 4. THE

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

PubMed

Ayyar, B Vijayalakshmi; Atassi, M Zouhair

2016-12-01

Binding behaviors of the H N and the H C domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H N and H C regions were prepared (H N 519-845 and H C 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrostatic Effects in Filamentous Protein Aggregation

PubMed Central

Buell, Alexander K.; Hung, Peter; Salvatella, Xavier; Welland, Mark E.; Dobson, Christopher M.; Knowles, Tuomas P.J.

2013-01-01

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules. PMID:23473495

Excited State Proton Transfer of Natural Flavonoids and Their Chromophores in Duplex and Tetraplex DNAs

PubMed Central

2015-01-01

Fisetin (3,7,3′,4′-tetrahydroxyflavone) and quercetin (3,5,7,3′,4′-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5′-d (CCCTAA-)n/(-TTAGGG)n-5′, two single-stranded DNA oligonucleotides, 5′-d(C3TA2)3C3-3′ and 5′-d(T2AG3)4-3′, and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid–DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own “reporter” for their interactions with macromolecular targets. PMID:25393681

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

PubMed Central

Tempelman, L A; Hammer, D A

1994-01-01

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 8 FIGURE 10 PMID:8038394

Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1.

PubMed

Sayre, M H; Geiduschek, E P

1990-08-01

The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

PubMed

Näreoja, Tuomas; Rosenholm, Jessica M; Lamminmäki, Urpo; Hänninen, Pekka E

2017-05-01

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L -1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Lectin functionalized ZnO nanoarrays as a 3D nano-biointerface for bacterial detection.

PubMed

Zheng, Laibao; Wan, Yi; Qi, Peng; Sun, Yan; Zhang, Dun; Yu, Liangmin

2017-05-15

The detection of pathogenic bacteria is essential in various fields, such as food safety, water environmental analysis, or clinical diagnosis. Although rapid and selective techniques have been achieved based on the fast and specific binding of recognitions elements and target, the sensitive detection of bacterial pathogens was limited by their low targets-binding efficiency. The three-dimensional (3D) nano-biointerface, compared with the two-dimensional (2D) flat substrate, has a much higher binding capacity, which can offer more reactive sites to bind with bacterial targets, resulting in a great improvement of detection sensitivity. Herein, a lectin functionalized ZnO nanorod (ZnO-NR) array has been fabricated and employed as a 3D nano-biointerface for Escherichia coli (E. coli) capture and detection by multivalent binding of concanavalin A (ConA) with polysaccharides on the cellular surface of E. coli. The 3D lectin functionalized ZnO-NR array-based assay shows reasonable detection limit and efficiently expanded linear range (1.0×10 3 to 1.0×10 7 cfumL -1 ) for pathogen detection. The platform has a potential for further applications and provides an excellent sensitivity approach for detection of pathogenic bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules.

PubMed

Zhao, Tao; Liu, Ran; Ding, Xiaofan; Zhao, Juncai; Yu, Haixiang; Wang, Lei; Xu, Qing; Wang, Xuan; Lou, Xinhui; He, Miao; Xiao, Yi

2015-08-04

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.

Direct protein detection with a nano-interdigitated array gate MOSFET.

PubMed

Tang, Xiaohui; Jonas, Alain M; Nysten, Bernard; Demoustier-Champagne, Sophie; Blondeau, Franoise; Prévot, Pierre-Paul; Pampin, Rémi; Godfroid, Edmond; Iñiguez, Benjamin; Colinge, Jean-Pierre; Raskin, Jean-Pierre; Flandre, Denis; Bayot, Vincent

2009-08-15

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.

Annexin A1 Complex Mediates Oxytocin Vesicle Transport

PubMed Central

Makani, Vishruti; Sultana, Rukhsana; Sie, Khin Sander; Orjiako, Doris; Tatangelo, Marco; Dowling, Abigail; Cai, Jian; Pierce, William; Butterfield, D. Allan; Hill, Jennifer; Park, Joshua

2013-01-01

Oxytocin is a major neuropeptide that modulates the brain functions involved in social behavior and interaction. Despite of the importance of oxytocin for neural control of social behavior, little is known about the molecular mechanism(s) by which oxytocin secretion in the brain is regulated. Pro-oxytocin is synthesized in the cell bodies of hypothalamic neurons in the supraoptic and paraventricular nuclei and processed to a 9-amino-acid mature form during post-Golgi transport to the secretion sites at the axon terminals and somatodendritic regions. Oxytocin secreted from the somatodendritic regions diffuses throughout the hypothalamus and its neighboring brain regions. Some oxytocin-positive axons innervate and secrete oxytocin to the brain regions distal to the hypothalamus. Brain oxytocin binds to its receptors in the brain regions involved in social behavior. Oxytocin is also secreted from the axon terminal at the posterior pituitary gland into the blood circulation. We have discovered a new molecular complex consisting of annexin A1 (ANXA1), A-kinase anchor protein 150 (AKAP150), and microtubule motor, that controls the distribution of oxytocin vesicles between the axon and the cell body in a protein kinase A (PKA)- and protein kinase C (PKC)-sensitive manner. ANXA1 showed significant co-localization with oxytocin vesicles. Activation of PKA enhanced the association of kinesin-2 with ANXA1, thus increasing the axon-localization of oxytocin vesicles. Conversely, activation of PKC decreased the binding of kinesin-2 to ANXA1, thus attenuating the axon-localization of oxytocin vesicles. Our study suggests that ANXA1 complex coordinates the actions of PKA and PKC to control the distribution of oxytocin vesicles between the axon and the cell body. PMID:24118254

Ropizine concurrently enhances and inhibits ( sup 3 H) dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

1990-01-01

Ropizine produces a simultaneous enhancement and inhibition of ({sup 3}H) dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity ({sup 3}H)DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances ({sup 3}H)DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+){minus} pentazocine, has not been fullymore » characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of ({sup 3}H)DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.« less

Biological Activity and Binding Site Characteristics of the PA1b Entomotoxin on Insects from Different Orders

PubMed Central

Gressent, Frédéric; Duport, Gabrielle; Rahioui, Isabelle; Pauchet, Yannick; Bolland, Patrice; Specty, Olivier; Rahbe, Yvan

2007-01-01

The aim of this work was to investigate both the biological activity of an entomotoxin, the pea albumin 1b (PA1b), and the presence or absence of its binding site within an array of insect species. The data obtained showed that insect sensitivity was not related to its taxonomic position. Moreover, PA1b was not toxic to several tested microorganisms. However, the binding site was found to be conserved among very different insects, displaying similar thermodynamic constants regardless of the in vivo species sensitivity. The binding site alone was, therefore, not sufficient for toxicity. One exception was the pea weevil, Bruchus pisorum, which was the only tested species without any detectable binding activity. These findings indicate that the binding site probably has an important endogenous function in insects and that adaptation to pea seeds resulted in the elimination of the toxin binding activity in two independent insect lineages. Other mechanisms are likely to interact with the toxin effects, although they are still largely unknown, but there is no evidence of any specific degradation of PA1b in the midgut of insects insensitive to the toxin, such as Drosophila melanogaster or Mamestra brassicae. PMID:20331395

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

PubMed

Voloshin, Olga; Gocheva, Yana; Gutnick, Marina; Movshovich, Natalia; Bakhrat, Anya; Baranes-Bachar, Keren; Bar-Zvi, Dudy; Parvari, Ruti; Gheber, Larisa; Raveh, Dina

2010-06-01

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

Behavioral Control and Reward Sensitivity in Adolescents' Risk Taking Behavior: A Longitudinal TRAILS Study.

PubMed

Peeters, Margot; Oldehinkel, Tineke; Vollebergh, Wilma

2017-01-01

Neurodevelopmental theories of risk behavior hypothesize that low behavioral control in combination with high reward sensitivity explains adolescents' risk behavior. However, empirical studies examining this hypothesis while including actual risk taking behavior in adolescence are lacking. In this study we tested whether the imbalance between behavioral control and reward sensitivity underlies risk taking behavior in adolescence, using a nationally representative longitudinal sample of 715 adolescents, of which 66% revealed an increased risk for mental health problems. To assess behavioral control at age 11 we used both self-report (effortful control) as well as behavioral measures of cognitive control (i.e., working memory and response inhibition). Reward sensitivity was assessed with the Bangor Gambling Task. The main finding of this study was that effortful control at age 11 was the best predictor of risk taking behavior (alcohol and cannabis use) at age 16, particularly among adolescents who were more reward sensitive. Risk taking behavior in adolescents might be explained by relatively weak behavioral control functioning combined with high sensitivity for reward.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, A.N.; Benson, S.C.

1998-07-21

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye. 10 figs.

A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

PubMed

Cinkornpumin, Jessica K; Wisidagama, Dona R; Rapoport, Veronika; Go, James L; Dieterich, Christoph; Wang, Xiaoyue; Sommer, Ralf J; Hong, Ray L

2014-10-15

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

DNA breathing dynamics distinguish binding from nonbinding consensus sites for transcription factor YY1 in cells.

PubMed

Alexandrov, Boian S; Fukuyo, Yayoi; Lange, Martin; Horikoshi, Nobuo; Gelev, Vladimir; Rasmussen, Kim Ø; Bishop, Alan R; Usheva, Anny

2012-11-01

The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.

Photoionization cross section and binding energy of single dopant in hollow cylindrical core/shell quantum dot

NASA Astrophysics Data System (ADS)

Feddi, E.; El-Yadri, M.; Dujardin, F.; Restrepo, R. L.; Duque, C. A.

2017-02-01

In this study, we have investigated the confined donor impurity in a hollow cylindrical-shell quantum dot. The charges are assumed to be completely confined to the interior of the shell with rigid walls. Within the framework of the effective-mass approximation and by using a simple variational approach, we have computed the donor binding energy as a function of the shell sizes in order to study the behavior of the electron-impurity attraction for a very small thickness. Our results show that the binding energy of a donor impurity placed at the center of cylindrical core/shell dots depends strongly on the shell size. The binding energy increases when the shell-wideness becomes smaller and shows the same behavior as in a simple cylindrical quantum dot. A special case has been studied, which corresponds to the ratio between the inner and outer radii near to one (a/b → 1) for which our model gives a non-significant behavior of the impurity binding energy. This fact implies the existence of a critical value (a/b) for which the binding energy of the donor impurity tends to the limit value of 4 effective Rydbergs as in a 2D quantum well. We also analyse the photoionization cross section considering only the in-plane incident radiation polarization. We determine its behavior as a function of photon energy, shell size, and donor position. The measurement of photoionization in such systems would be of great interest to understand the optical properties of carriers in quantum dots.

Lipid A binding proteins in macrophages detected by ligand blotting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

1987-05-01

Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

PubMed

Cinar, Süleyman; Al-Ayoubi, Samy; Sternemann, Christian; Peters, Judith; Winter, Roland; Czeslik, Claus

2018-01-31

Calmodulin (CaM) is a Ca 2+ sensor and mediates Ca 2+ signaling through binding of numerous target ligands. The binding of ligands by Ca 2+ -saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

PubMed

Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés D; Kuroda, Shun'ichi

2016-06-01

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The binding of analogs of porphyrins and chlorins with elongated side chains to albumin

PubMed Central

Ben Dror, Shimshon; Bronshtein, Irena; Weitman, Hana; Smith, Kevin M.; O’Neal, William G.; Jacobi, Peter A.; Ehrenberg, Benjamin

2012-01-01

In previous studies, we demonstrated that elongation of side chains of several sensitizers endowed them with higher affinity for artificial and natural membranes and caused their deeper localization in membranes. In the present study, we employed eight hematoporphyrin and protoporphyrin analogs and four groups containing three chlorin analogs each, all synthesized with variable numbers of methylenes in their alkyl carboxylic chains. We show that these tetrapyrroles’ affinity for bovine serum albumin (BSA) and their localization in the binding site are also modulated by chain lengths. The binding constants of the hematoporphyrins and protoporphyrins to BSA increased as the number of methylenes was increased. The binding of the chlorins depended on the substitution at the meso position opposite to the chains. The quenching of the sensitizers’ florescence by external iodide ions decreased as the side chains became longer, indicating to deeper insertion of the molecules into the BSA binding pocket. To corroborate this conclusion, we studied the efficiency of photodamage caused to tryptophan in BSA upon illumination of the bound sensitizers. The efficiency was found to depend on the side-chain lengths of the photosensitizer. We conclude that the protein site that hosts these sensitizers accommodates different analogs at positions that differ slightly from each other. These differences are manifested in the ease of access of iodide from the external aqueous phase, and in the proximity of the photosensitizers to the tryptophan. In the course of this study, we developed the kinetic equations that have to be employed when the sensitizer itself is being destroyed. PMID:19330323

A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murtaugh, Megan L.; Fanning, Sean W.; Sharma, Tressa M.

2012-09-05

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK{sub a} change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novelmore » combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine 'hot-spots,' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.« less

Motor-substrate interactions in mycoplasma motility explains non-Arrhenius temperature dependence.

PubMed

Chen, Jing; Neu, John; Miyata, Makoto; Oster, George

2009-12-02

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by approximately 400 "leg" proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40 degrees C. This corresponds to an Arrhenius factor that decreases from approximately 45 k(B)T at 10 degrees C to approximately 10 k(B)T at 40 degrees C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.

Motor-Substrate Interactions in Mycoplasma Motility Explains Non-Arrhenius Temperature Dependence

PubMed Central

Chen, Jing; Neu, John; Miyata, Makoto; Oster, George

2009-01-01

Abstract Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by ∼400 “leg” proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10–40°C. This corresponds to an Arrhenius factor that decreases from ∼45 kBT at 10°C to ∼10 kBT at 40°C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction. PMID:19948122

Comparison of mHTT Antibodies in Huntington’s Disease Mouse Models Reveal Specific Binding Profiles and Steady-State Ubiquitin Levels with Disease Development

PubMed Central

Bayram-Weston, Zubeyde; Jones, Lesley; Dunnett, Stephen B.; Brooks, Simon P.

2016-01-01

Huntington’s disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. PMID:27196694

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

Exo-Dye-based assay for rapid, inexpensive, and sensitive detection of DNA-binding proteins.

PubMed

Chen, Zaozao; Ji, Meiju; Hou, Peng; Lu, Zuhong

2006-07-07

We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.

In Vivo [11C]Dihydrotetrabenazine ([11C]DTBZ) Binding in Rat Striatum: Sensitivity to Dopamine Concentrations

PubMed Central

Kilbourn, Michael R.; Butch, Elizabeth R.; Desmond, Timothy; Sherman, Phillip; Harris, Paul E.; Frey, Kirk A.

2009-01-01

Introduction The sensitivity of the in vivo binding of [11C]dihydrotetrabenazine ([11C]DTBZ) and [11C]methylphenidate ([11C]MPH) to their respective targets, the vesicular monoamine transporter (VMAT2) and the neuronal membrane dopamine transporter (DAT), after alterations of endogenous levels of dopamine were examined in the rat brain. Methods In vivo binding of [11C]DTBZ and [11C]MPH were determined using a bolus+infusion protocol. In vitro numbers of VMAT2 binding sites were determined by autoradiography. Results Repeated dosing with α-methyl-p-tyrosine (AMPT) at doses that significantly (−75%) depleted brain tissue dopamine levels resulted in increased (+36%) in vivo [11C]DTBZ binding to VMAT2 in the striatum. The increase in binding could be completely reversed by treatment with L-DOPA/benserazide to restore dopamine levels. There were no changes in total numbers of VMAT2 binding sites as measured using in vitro autoradiography. No changes were observed for in vivo [11C]MPH binding to the DAT in the striatum following AMPT pretreatment. Conclusion These results indicate that large reductions of dopamine concentrations in the rat brain can produce modest but significant changes in binding of radioligands to the VMAT2, which can be reversed by repleneshment of dopamine using exogenous L-DOPA. PMID:20122661

T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFβ dosage.

PubMed Central

Talebian, Laleh; Li, Zhe; Guo, Yalin; Gaudet, Justin; Speck, Maren E.; Sugiyama, Daisuke; Kaur, Prabhjot; Pear, Warren S.; Maillard, Ivan; Speck, Nancy A.

2007-01-01

The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non–DNA-binding partner CBFβ. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBFβ levels by 3- or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBFβ levels, and were virtually absent when CBFβ levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4−CD8− double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBFβ threshold for normal T-cell development, and situate an essential role for core-binding factors during the earliest stages of T-cell development. PMID:16940420

Getting what you want: How fit between desired and received leader sensitivity influences emotion and counterproductive work behavior.

PubMed

Rupprecht, Elizabeth A; Kueny, Clair Reynolds; Shoss, Mindy K; Metzger, Andrew J

2016-10-01

We challenge the intuitive belief that greater leader sensitivity is always associated with desirable outcomes for employees and organizations. Specifically, we argue that followers' idiosyncratic desires for, and perceptions of, leader sensitivity behaviors play a key role in how followers react to their leader's sensitivity. Moreover, these resulting affective experiences are likely to have important consequences for organizations, specifically as they relate to employee counterproductive work behavior (CWB). Drawing from supplies-values (S-V) fit theory and the stressor-emotion model of CWB, the current study focuses on the affective and behavioral consequences of fit between subordinates' ideal leader sensitivity behavior preferences and subordinates' perceptions of their actual leader's sensitivity behaviors. Polynomial regression analyses reveal that congruence between ideal and actual leader sensitivity influences employee negative affect and, consequently, engagement in counterproductive work behavior. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

PubMed Central

Han, Xue-Sheng; Dahlquist, Frederick W.; Parkinson, John S.

2017-01-01

A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON–OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands. PMID:28827352

Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

PubMed

Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

2016-01-01

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

Autonomic nervous system function, child behavior, and maternal sensitivity in three-year-old children with surgically corrected transposition.

PubMed

Harrison, Tondi M

2013-01-01

Explore relationships among autonomic nervous system (ANS) function, child behavior, and maternal sensitivity in three-year-old children with surgically corrected transposition of the great arteries (TGA) and in children healthy at birth. Children surviving complex congenital heart defects are at risk for behavior problems. ANS function is associated with behavior and with maternal sensitivity. Child ANS function (heart rate variability) and maternal sensitivity (Parent-Child Early Relational Assessment) were measured during a challenging task. Mother completed the Child Behavior Checklist. Data were analyzed descriptively and graphically. Children with TGA had less responsive autonomic function and more behavior problems than healthy children. Autonomic function improved with more maternal sensitivity. Alterations in ANS function may continue years after surgical correction in children with TGA, potentially impacting behavioral regulation. Maternal sensitivity may be associated with ANS function in this population. Continued research on relationships among ANS function, child behavior, and maternal sensitivity is warranted. Copyright © 2013 Elsevier Inc. All rights reserved.

Putative calcium-binding domains of the Caenorhabditis elegans BK channel are dispensable for intoxication and ethanol activation

PubMed Central

Davis, S. J.; Scott, L. L.; Ordemann, G.; Philpo, A.; Cohn, J.; Pierce-Shimomura, J. T.

2016-01-01

Alcohol modulates the highly conserved, voltage- and calcium-activated potassium (BK) channel, which contributes to alcohol-mediated behaviors in species from worms to humans. Previous studies have shown that the calcium-sensitive domains, RCK1 and the Ca2+ bowl, are required for ethanol activation of the mammalian BK channel in vitro. In the nematode Caenorhabditis elegans, ethanol activates the BK channel in vivo, and deletion of the worm BK channel, SLO-1, confers strong resistance to intoxication. To determine if the conserved RCK1 and calcium bowl domains were also critical for intoxication and basal BK channel-dependent behaviors in C. elegans, we generated transgenic worms that express mutated SLO-1 channels predicted to have the RCK1, Ca2+ bowl or both domains rendered insensitive to calcium. As expected, mutating these domains inhibited basal function of SLO-1 in vivo as neck and body curvature of these mutants mimicked that of the BK null mutant. Unexpectedly, however, mutating these domains singly or together in SLO-1 had no effect on intoxication in C. elegans. Consistent with these behavioral results, we found that ethanol activated the SLO-1 channel in vitro with or without these domains. By contrast, in agreement with previous in vitro findings, C. elegans harboring a human BK channel with mutated calcium-sensing domains displayed resistance to intoxication. Thus, for the worm SLO-1 channel, the putative calcium-sensitive domains are critical for basal in vivo function but unnecessary for in vivo ethanol action. PMID:26113050

Comparative Effects of Oral Chlorpyrifos Exposure on Cholinesterase Activity and Muscarinic Receptor Binding in Neonatal and Adult Rat Heart

PubMed Central

Howard, Marcia D.; Mirajkar, Nikita; Karanth, Subramanya; Pope, Carey N.

2010-01-01

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M2 muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M2 receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor–mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M2 receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac ChE activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1 × LD10: neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1 × LD10, relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (≈ 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC50 values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82–90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4 hours after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity. PMID:17644233

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1994-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a polycationic chain of at least two positive charges. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Loss of selenium-binding protein 1 decreases sensitivity to clastogens and intracellular selenium content in HeLa cells

USDA-ARS?s Scientific Manuscript database

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesized that loss of SBP1 modulates cellular selenium content and the response of ...

Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

ERIC Educational Resources Information Center

Nixon, J. E.

1977-01-01

Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

Studying red blood cell agglutination by measuring membrane viscosity with optical tweezers

NASA Astrophysics Data System (ADS)

Fernandes, Heloise P.; Fontes, Adriana; de Thomaz, André A.; Barbosa, Luiz C.; Barjas-Castro, Maria L.; Cesar, Carlos L.

2007-09-01

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.

Proton NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertini, I.; Luchinat, C.; Messori, L.

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N/sub 3//sup -/, NCO/sup -/, and NCS/sup -/) has been studied by /sup 1/H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E/times/I, E/times/I/sub 2/, and E/times/I/times/X, each with characteristic NMR features, have been identified. The NMR data suggest that when the carboxylate group of a substrate of inhibitor binds atmore » the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The /sup 1/H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes. These signals are relatively constant for all E/times/I and E/times/I/times/X complexes, indicating that this ligand is in a relatively fixed or buried conformation. However in the 2:1 carboxylate inhibitor (E/times/I/sub 2/) complexes, both signals are shifted upfield, suggesting a disturbance in the interaction of this histidine with the metal.« less

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

PubMed

Pany, Satyabrata; Ghosh, Anamitra; You, Youngki; Nguyen, Nga; Das, Joydip

2017-11-01

Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca 2+ -sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca 2+ -sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1 H567K , a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice

PubMed Central

2012-01-01

Background Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. Results The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. Conclusions These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior. PMID:23268962

Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice.

PubMed

Nagura, Hitoshi; Ishikawa, Yasuyuki; Kobayashi, Katsunori; Takao, Keizo; Tanaka, Tomo; Nishikawa, Kouki; Tamura, Hideki; Shiosaka, Sadao; Suzuki, Hidenori; Miyakawa, Tsuyoshi; Fujiyoshi, Yoshinori; Doi, Tomoko

2012-12-26

Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.

BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain.

PubMed

Hagn, Franz; Klein, Christian; Demmer, Oliver; Marchenko, Natasha; Vaseva, Angelina; Moll, Ute M; Kessler, Horst

2010-01-29

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

Decreased expression of extracellular matrix proteins and trophic factors in the amygdala complex of depressed mice after chronic immobilization stress

PubMed Central

2012-01-01

Background The amygdala plays an essential role in controlling emotional behaviors and has numerous connections to other brain regions. The functional role of the amygdala has been highlighted by various studies of stress-induced behavioral changes. Here we investigated gene expression changes in the amygdala in the chronic immobilization stress (CIS)-induced depression model. Results Eight genes were decreased in the amygdala of CIS mice, including genes for neurotrophic factors and extracellular matrix proteins. Among these, osteoglycin, fibromodulin, insulin-like growth factor 2 (Igf2), and insulin-like growth factor binding protein 2 (Igfbp2) were further analyzed for histological expression changes. The expression of osteoglycin and fibromodulin simultaneously decreased in the medial, basolateral, and central amygdala regions. However, Igf2 and Igfbp2 decreased specifically in the central nucleus of the amygdala. Interestingly, this decrease was found only in the amygdala of mice showing higher immobility, but not in mice displaying lower immobility, although the CIS regimen was the same for both groups. Conclusions These results suggest that the responsiveness of the amygdala may play a role in the sensitivity of CIS-induced behavioral changes in mice. PMID:22672618

Optimizing pH response of affinity between protein G and IgG Fc: how electrostatic modulations affect protein-protein interactions.

PubMed

Watanabe, Hideki; Matsumaru, Hiroyuki; Ooishi, Ayako; Feng, Yanwen; Odahara, Takayuki; Suto, Kyoko; Honda, Shinya

2009-05-01

Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by "enthalpy-entropy compensation." Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.

Positron Emission Tomography of Brain β-Amyloid and Tau Levels in Adults With Down Syndrome

PubMed Central

Nelson, Linda D.; Siddarth, Prabha; Kepe, Vladimir; Scheibel, Kevin E.; Huang, S. C.; Barrio, Jorge R.; Small, Gary W.

2012-01-01

Objectives To determine the neuropathological load in the living brain of nondemented adults with Down syndrome using positron emission tomography with 2-(1-{6-[(2-fluorine 18–labeled fluoroethyl)methylamino]-2-napthyl}ethylidene) malononitrile ([18F]FDDNP) and to assess the influence of age and cognitive and behavioral functioning. For reference, [18F]FDDNP binding values and patterns were compared with those from patients with Alzheimer disease and cognitively intact control participants. Design Cross-sectional clinical study. Participants Volunteer sample of 19 persons with Down syndrome without dementia (mean age, 36.7 years), 10 patients with Alzheimer disease (mean age, 66.5 years), and 10 controls (mean age, 43.8 years). Main Outcome Measures Binding of [18F]FDDNP in brain regions of interest, including the parietal, medial temporal, lateral temporal, and frontal lobes and posterior cingulate gyrus, and the average of all regions (global binding). Results The [18F]FDDNP binding values were higher in all brain regions in the Down syndrome group than in controls. Compared with the Alzheimer disease group, the Down syndrome group had higher [18F]FDDNP binding values in the parietal and frontal regions, whereas binding levels in other regions were comparable. Within the Down syndrome group, age correlated with [18F]FDDNP binding values in all regions except the posterior cingulate, and several measures of behavioral dysfunction showed positive correlations with global, frontal, parietal, and posterior cingulate [18F]FDDNP binding. Conclusions Consistent with neuropathological findings from postmortem studies, [18F]FDDNP positron emission tomography shows high binding levels in Down syndrome comparable to Alzheimer disease and greater levels than in members of a control group. The positive associations between [18F]FDDNP binding levels and age as well as behavioral dysfunction in Down syndrome are consistent with the age-related progression of Alzheimer-type neuropathological findings in this population. PMID:21670401

Sensitive and accurate identification of protein–DNA binding events in ChIP-chip assays using higher order derivative analysis

PubMed Central

Barrett, Christian L.; Cho, Byung-Kwan

2011-01-01

Immuno-precipitation of protein–DNA complexes followed by microarray hybridization is a powerful and cost-effective technology for discovering protein–DNA binding events at the genome scale. It is still an unresolved challenge to comprehensively, accurately and sensitively extract binding event information from the produced data. We have developed a novel strategy composed of an information-preserving signal-smoothing procedure, higher order derivative analysis and application of the principle of maximum entropy to address this challenge. Importantly, our method does not require any input parameters to be specified by the user. Using genome-scale binding data of two Escherichia coli global transcription regulators for which a relatively large number of experimentally supported sites are known, we show that ∼90% of known sites were resolved to within four probes, or ∼88 bp. Over half of the sites were resolved to within two probes, or ∼38 bp. Furthermore, we demonstrate that our strategy delivers significant quantitative and qualitative performance gains over available methods. Such accurate and sensitive binding site resolution has important consequences for accurately reconstructing transcriptional regulatory networks, for motif discovery, for furthering our understanding of local and non-local factors in protein–DNA interactions and for extending the usefulness horizon of the ChIP-chip platform. PMID:21051353

An electrochemical sensing platform based on local repression of electrolyte diffusion for single-step, reagentless, sensitive detection of a sequence-specific DNA-binding protein.

PubMed

Zhang, Yun; Liu, Fang; Nie, Jinfang; Jiang, Fuyang; Zhou, Caibin; Yang, Jiani; Fan, Jinlong; Li, Jianping

2014-05-07

In this paper, we report for the first time an electrochemical biosensor for single-step, reagentless, and picomolar detection of a sequence-specific DNA-binding protein using a double-stranded, electrode-bound DNA probe terminally modified with a redox active label close to the electrode surface. This new methodology is based upon local repression of electrolyte diffusion associated with protein-DNA binding that leads to reduction of the electrochemical response of the label. In the proof-of-concept study, the resulting electrochemical biosensor was quantitatively sensitive to the concentrations of the TATA binding protein (TBP, a model analyte) ranging from 40 pM to 25.4 nM with an estimated detection limit of ∼10.6 pM (∼80 to 400-fold improvement on the detection limit over previous electrochemical analytical systems).

Correlation between the enhancement of flunitrazepam binding by GABA and seizure susceptibility in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marley, R.J.; Wehner, J.M.

Various populations of mice exhibit differential sensitivity to seizure-inducing agents. The relationship of seizure susceptibility to alterations in the GABA receptor complex was investigated in six different populations of mice consisting of four inbred strains (C57BL, DBA, C3H, and BALB) and two selected lines (long sleep and short sleep). Seizure activity was induced by intraperitoneal administration of the GAD inhibitor, 3-mercaptopropionic acid, and latencies to seizure onset and tonus were measured. In naive mice of the same populations, GABA enhancement of TH-flunitrazepam binding was measured in extensively washed whole brain membranes at several GABA concentrations. Both differential seizure sensitivity tomore » 3-mercaptopropionic acid and differential enhancement of TH-flunitrazepam binding by GABA were observed in these six populations of mice. Correlational analyses indicated a positive correlation between the degree of GABA enhancement of TH-flunitrazepam binding and resistance to the seizure-inducing properties of 3-mercaptopropionic acid. These data suggest that genetic differences in sensitivity to seizure-inducing agents that disrupt the GABAergic system may be related to differences in coupling between the various receptors associated with the GABA receptor complex.« less

[11C]Flumazenil PET in patients with epilepsy with dual pathology.

PubMed

Juhász, C; Nagy, F; Muzik, O; Watson, C; Shah, J; Chugani, H T

1999-05-01

Coexistence of hippocampal sclerosis and a potentially epileptogenic cortical lesion is referred to as dual pathology and can be responsible for poor surgical outcome in patients with medically intractable partial epilepsy. [11C]Flumazenil (FMZ) positron emission tomography (PET) is a sensitive method for visualizing epileptogenic foci. In this study of 12 patients with dual pathology, we addressed the sensitivity of FMZ PET to detect hippocampal abnormalities and compared magnetic resonance imaging (MRI) with visual as well as quantitative FMZ PET findings. All patients underwent volumetric MRI, prolonged video-EEG monitoring, and glucose metabolism PET before the FMZ PET. MRI-coregistered partial volume-corrected PET images were used to measure FMZ-binding asymmetries by using asymmetry indices (AIs) in the whole hippocampus and in three (anterior, middle, and posterior) hippocampal subregions. Cortical sites of decreased FMZ binding also were evaluated by using AIs for regions with MRI-verified cortical lesions as well as for non-lesional areas with visually detected asymmetry. Abnormally decreased FMZ binding could be detected by quantitative analysis in the atrophic hippocampus of all 12 patients, including three patients with discordant or inconclusive EEG findings. Decreased FMZ binding was restricted to only one subregion of the hippocampus in three patients. Areas of decreased cortical FMZ binding were obvious visually in all patients. Decreased FMZ binding was detected visually in nonlesional cortical areas in four patients. The AIs for these nonlesional regions with visual asymmetry were significantly lower than those for regions showing MRI lesions (paired t test, p = 0.0075). Visual as well as quantitative analyses of FMZ-binding asymmetry are sensitive methods to detect decreased benzodiazepine-receptor binding in the hippocampus and neocortex of patients with dual pathology. MRI-defined hippocampal atrophy is always associated with decreased FMZ binding, although the latter may be localized to only one sub-region within the hippocampus. FMZ PET abnormalities can occur in areas with normal appearance on MRI, but FMZ-binding asymmetry of these regions is lower when compared with that of lesional areas. FMZ PET can be especially helpful when MRI and EEG findings of patients with intractable epilepsy are discordant.

Dynamics simulations for engineering macromolecular interactions

NASA Astrophysics Data System (ADS)

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Dynamics simulations for engineering macromolecular interactions.

PubMed

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20,000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Relationship between L-DOPA-induced reduction in motor and exploratory activity and degree of DAT binding in the rat

PubMed Central

Nikolaus, Susanne; Beu, Markus; De Souza Silva, Angelica Maria; Huston, Joseph P.; Hautzel, Hubertus; Chao, Owen Y.; Antke, Christina; Müller, Hans-Wilhelm

2014-01-01

Purpose: The present study assessed the influence of L-DOPA administration on neostriatal dopamine (DA) transporter (DAT) binding in relation to motor and exploratory behaviors in the rat. Methods: Rats received injections of 5 mg/kg L-DOPA, 10 mg/kg L-DOPA or vehicle. Motor and exploratory behaviors were assessed for 30 min in an open field prior to administration of [123I]FP-CIT. Dopamine transporter binding was measured with small animal single-photon emission computed tomography (SPECT) 2 h after radioligand administration for 60 min. Results: Both L-DOPA doses significantly reduced DAT binding and led to significantly less head-shoulder motility and more sitting relative to vehicle. Moreover, 10 mg/kg L-DOPA induced less distance traveled and ambulation than 5 mg/kg L-DOPA. Analysis of time-behavior (t-b) curves showed that L-DOPA-treated animals relative to vehicle exhibited (1) a faster rate of increase in duration of sitting; (2) a slower rate of increase in duration of head-shoulder motility; and (3) a slower rate of decrease in frequency of head-shoulder motility. Conclusions: The reductions of striatal DAT binding after L-DOPA challenges reflected elevated concentrations of synaptic DA. L-DOPA-treated animals showed less head-shoulder motility and more sitting than vehicle-treated animals, indicating an association between less behavioral activity and increased availability of striatal DA. The faster increase of sitting duration to a higher final level and the slower increase of head-shoulder motility to a lower final level relative to controls may be interpreted in terms on behavioral habituation to a novel environment. PMID:25566000

Longitudinal imaging of the availability of dopamine transporter and D2 receptor in rat striatum following mild ischemia.

PubMed

Momosaki, Sotaro; Ito, Miwa; Yamato, Hiroko; Iimori, Hitoshi; Sumiyoshi, Hirokazu; Morimoto, Kenji; Imamoto, Natsumi; Watabe, Tadashi; Shimosegawa, Eku; Hatazawa, Jun; Abe, Kohji

2017-02-01

The changes in the availability of striatal dopamine transporter and dopamine D2 receptor after mild focal ischemia in rats were measured using a small animal positron emission tomography system. Mild focal ischemia was induced by 20-minute middle cerebral artery occlusion. [ 11 C]PE2I binding to dopamine transporter was transiently increased on the ipsilateral side of the striatum at 2 days after middle cerebral artery occlusion. On day 7 and 14 after middle cerebral artery occlusion, [ 11 C]PE2I binding levels were decreased. In contrast, [ 11 C]raclopride binding to dopamine D2 receptor in the ipsilateral striatum had not changed at 2 days after middle cerebral artery occlusion. [ 11 C]Raclopride binding was significantly decreased on the ischemic side of the striatum at 7 and 14 days after middle cerebral artery occlusion. Moreover, on day 1 and 2 after middle cerebral artery occlusion, significant circling behavior to the contralateral direction was induced by amphetamine challenge. This behavior disappeared at 7 days after middle cerebral artery occlusion. At 14 days, circling behavior to the ipsilateral direction (middle cerebral artery occlusion side) was significantly increased, and that to the contralateral direction also appeared again. The present study suggested that amphetamine-induced circling behavior indicated striatal dopaminergic alterations and that dopamine transporter and dopamine D2 receptor binding could be key markers for predicting motor dysfunction after mild focal ischemia.

Odorant-binding proteins display high affinities for behavioral attractants and repellents in the natural predator Chrysopa pallens.

PubMed

Li, Zhao-Qun; Zhang, Shuai; Luo, Jun-Yu; Wang, Si-Bao; Dong, Shuang-Lin; Cui, Jin-Jie

2015-07-01

Chrysopa pallens is an important natural predator of various pests in many different cropping systems. Understanding the sophisticated olfactory system of insect antennae is crucial for studying the physiological bases of olfaction and could also help enhance the effectiveness of C. pallens in biological control. However, functional studies of the olfactory genes in C. pallens are still lacking. In this study, we cloned five odorant-binding protein (OBP) genes from C. pallens (CpalOBPs). Quantitative RT-PCR results indicated that the five CpalOBPs had different tissue expression profiles. Ligand-binding assays showed that farnesol, farnesene, cis-3-hexenyl hexanoate, geranylacetone, beta-ionone, octyl aldehyde, decanal, nerolidol (Ki<20 μM), and especially 2-pentadecanone (Ki=1.19 μM) and 2-hexyl-1-decanol (Ki=0.37 μM) strongly bound to CpalOBP2. CpalOBP15 exhibited high binding affinities for beta-ionone, 2-tridecanone, trans-nerolidol, and dodecyl aldehyde. Behavioral trials using the 14 compounds exhibiting high binding affinities for the CpalOBPs revealed that nine were able to elicit significant behavioral responses from C. pallens. Among them, farnesene and its corresponding alcohol, farnesol, elicited remarkable repellent behavioral responses from C. pallens. Our study provides several compounds that could be selected to develop slow-release agents that attract/repel C. pallens and to improve the search for strategies to eliminate insect pests. Copyright © 2015 Elsevier Inc. All rights reserved.

Neural correlates of behavioral amplitude modulation sensitivity in the budgerigar midbrain

PubMed Central

Neilans, Erikson G.; Abrams, Kristina S.; Idrobo, Fabio; Carney, Laurel H.

2016-01-01

Amplitude modulation (AM) is a crucial feature of many communication signals, including speech. Whereas average discharge rates in the auditory midbrain correlate with behavioral AM sensitivity in rabbits, the neural bases of AM sensitivity in species with human-like behavioral acuity are unexplored. Here, we used parallel behavioral and neurophysiological experiments to explore the neural (midbrain) bases of AM perception in an avian speech mimic, the budgerigar (Melopsittacus undulatus). Behavioral AM sensitivity was quantified using operant conditioning procedures. Neural AM sensitivity was studied using chronically implanted microelectrodes in awake, unrestrained birds. Average discharge rates of multiunit recording sites in the budgerigar midbrain were insufficient to explain behavioral sensitivity to modulation frequencies <100 Hz for both tone- and noise-carrier stimuli, even with optimal pooling of information across recording sites. Neural envelope synchrony, in contrast, could explain behavioral performance for both carrier types across the full range of modulation frequencies studied (16–512 Hz). The results suggest that envelope synchrony in the budgerigar midbrain may underlie behavioral sensitivity to AM. Behavioral AM sensitivity based on synchrony in the budgerigar, which contrasts with rate-correlated behavioral performance in rabbits, raises the possibility that envelope synchrony, rather than average discharge rate, might also underlie AM perception in other species with sensitive AM detection abilities, including humans. These results highlight the importance of synchrony coding of envelope structure in the inferior colliculus. Furthermore, they underscore potential benefits of devices (e.g., midbrain implants) that evoke robust neural synchrony. PMID:26843608

Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics

PubMed Central

Bianchi, Paola; Fermo, Elisa; Vercellati, Cristina; Marcello, Anna P.; Porretti, Laura; Cortelezzi, Agostino; Barcellini, Wilma; Zanella, Alberto

2012-01-01

Background The laboratory diagnosis of hereditary spherocytosis commonly relies on NaCl-based or glycerol-based red cell osmotic fragility tests; more recently, an assay directly targeting the hereditary spherocytosis molecular defect (eosin-5′-maleimide-binding test) has been proposed. None of the available tests identifies all cases of hereditary spherocytosis. Design and Methods We compared the performances of the eosin-5′-maleimide-binding test, NaCl-osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, and the Pink test on a series of 150 patients with hereditary spherocytosis grouped according to clinical phenotype and the defective protein, with the final aim of finding the combination of tests associated with the highest diagnostic power, even in the mildest cases of hereditary spherocytosis. Results The eosin-5′-maleimide-binding test had a sensitivity of 93% and a specificity of 98% for detecting hereditary spherocytosis: the sensitivity was independent of the type and amount of molecular defect and of the clinical phenotype. The acidified glycerol lysis test and Pink test showed comparable sensitivity (95% and 91%). The sensitivity of NaCl osmotic fragility tests, commonly considered the gold standard for the diagnosis of hereditary spherocytosis, was 68% on fresh blood and 81% on incubated blood, and further decreased in compensated cases (53% and 64%, respectively). The combination of the eosin-5′-maleimide-binding test and acidified glycerol lysis test enabled all patients with hereditary spherocytosis to be identified. The eosin-5′-maleimide-binding test showed the greatest disease specificity. Conclusions Each type of test fails to diagnose some cases of hereditary spherocytosis. The association of an eosin-5′-maleimide-binding test and an acidified glycerol lysis test enabled identification of all patients with hereditary spherocytosis in this series and, therefore, represents a currently effective diagnostic strategy for hereditary spherocytosis including mild/compensated cases. PMID:22058213

Estrogen Receptor-β Agonist Diarylpropionitrile: Biological Activities of R- and S-Enantiomers on Behavior and Hormonal Response to Stress

PubMed Central

Weiser, Michael J.; Wu, T. John; Handa, Robert J.

2009-01-01

Estrogens have been shown to have positive and negative effects on anxiety and depressive-like behaviors, perhaps explained by the existence of two distinct estrogen receptor (ER) systems, ERα and ERβ. The ERβ agonist, diarylpropionitrile (DPN) has been shown to have anxiolytic properties in rats. DPN exists as a racemic mixture of two enantiomers, R-DPN and S-DPN. In this study, we compared R-DPN and S-DPN for their in vitro binding affinity, ability to activate transcription in vitro at an estrogen response element, and in vivo endocrine and behavioral responses. In vitro binding studies using recombinant rat ERβ revealed that S-DPN has a severalfold greater relative binding affinity for ERβ than does R-DPN. Furthermore, cotransfection of N-38 immortalized hypothalamic cells with an estrogen response element-luc reporter and ERβ revealed that S-DPN is a potent activator of transcription in vitro, whereas R-DPN is not. Subsequently, we examined anxiety-like behaviors using the open-field test and elevated plus maze or depressive-like behaviors, using the forced swim test. Ovariectomized young adult female Sprague Dawley rats treated with racemic DPN, S-DPN, and the ERβ agonist, WAY-200070, showed significantly decreased anxiety-like behaviors in both the open-field and elevated plus maze and significantly less depressive-like behaviors in the forced swim test compared with vehicle-, R-DPN-, or propylpyrazoletriol (ERα agonist)-treated animals. In concordance with the relative binding affinity and transcriptional potency, these results demonstrate that the S-enantiomer is the biologically active form of DPN. These studies also indicate that estrogen's positive effects on mood, including its anxiolytic and antidepressive actions, are due to its actions at ERβ. PMID:19074580

Behavioral and Electrophysiological Correlates of Memory Binding Deficits in Patients at Different Risk Levels for Alzheimer's Disease.

PubMed

Pietto, Marcos; Parra, Mario A; Trujillo, Natalia; Flores, Facundo; García, Adolfo M; Bustin, Julian; Richly, Pablo; Manes, Facundo; Lopera, Francisco; Ibáñez, Agustín; Baez, Sandra

2016-06-30

Deficits in visual short-term memory (VSTM) binding have been proposed as an early and specific marker for Alzheimer's disease (AD). However, no studies have explored the neural correlates of this domain in clinical categories involving prodromal stages with different risk levels of conversion to AD. We assessed underlying electrophysiological modulations in patients with mild cognitive impairment (MCI), patients in the MCI stages of familial AD carrying the mutation E280A of the presenilin-1 gene (MCI-FAD), and healthy controls. Moreover, we compared the behavioral performance and neural correlates of both patient groups. Participants completed a change-detection VSTM task assessing recognition of changes between shapes or shape-color bindings, presented in two consecutive arrays (i.e., study and test) while event related potentials (ERPs) were recorded. Changes always occurred in the test array and consisted of new features replacing studied features (shape-only) or features swapping across items (shape-color binding). Both MCI and MCI-FAD patients performed worse than controls in the shape-color binding condition. Early electrophysiological activity (100-250 ms) was significantly reduced in both clinical groups, particularly over fronto-central and parieto-occipital regions. However, shape-color binding performance and their reduced neural correlates were similar between MCI and MCI-FAD. Our results support the validity of the VSTM binding test and their neural correlates in the early detection of AD and highlight the importance of studies comparing samples at different risk for AD conversion. The combined analysis of behavioral and ERP data gleaned with the VSTM binding task can offer a valuable memory biomarker for AD.

Highly sensitive hydrogen sulfide (H2 S) gas sensors from viral-templated nanocrystalline gold nanowires

NASA Astrophysics Data System (ADS)

Moon, Chung Hee; Zhang, Miluo; Myung, Nosang V.; Haberer, Elaine D.

2014-04-01

A facile, site-specific viral-templated assembly method was used to fabricate sensitive hydrogen sulfide (H2S) gas sensors at room temperature. A gold-binding M13 bacteriophage served to organize gold nanoparticles into linear arrays which were used as seeds for subsequent nanowire formation through electroless deposition. Nanowire widths and densities within the sensors were modified by electroless deposition time and phage concentration, respectively, to tune device resistance. Chemiresistive H2S gas sensors with superior room temperature sensing performance were produced with sensitivity of 654%/ppmv, theoretical lowest detection limit of 2 ppbv, and 70% recovery within 9 min for 0.025 ppmv. The role of the viral template and associated gold-binding peptide was elucidated by removing organics using a short O2 plasma treatment followed by an ethanol dip. The template and gold-binding peptide were crucial to electrical and sensor performance. Without surface organics, the resistance fell by several orders of magnitude, the sensitivity dropped by more than a factor of 100 to 6%/ppmv, the lower limit of detection increased, and no recovery was detected with dry air flow. Viral templates provide a novel, alternative fabrication route for highly sensitive, nanostructured H2S gas sensors.

Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy.

PubMed

Wang, Kun; Fan, Daoqing; Liu, Yaqing; Wang, Erkang

2015-11-15

Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of non-cancer cells and different cancer cells (10(4) cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 10(5) cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzyme-free and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy*

PubMed Central

Neundlinger, Isabel; Puntheeranurak, Theeraporn; Wildling, Linda; Rankl, Christian; Wang, Lai-Xi; Gruber, Hermann J.; Kinne, Rolf K. H.; Hinterdorfer, Peter

2014-01-01

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2′-aminoethyl β-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. PMID:24962566

Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

PubMed Central

Sytnikova, Yuliya A.; Kubarenko, Andriy V.; Schäfer, Andrea; Weber, Alexander N. R.; Niehrs, Christof

2011-01-01

Background The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids. Principal Findings Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function. Significance The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle. PMID:21249130

L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry

NASA Technical Reports Server (NTRS)

Holstein, G. R.; Martinelli, G. P.; Cohen, B.

1992-01-01

L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

Dioxin Sensitivity-Related Two Critical Amino Acids of Arylhydrocarbon Receptor May Not Correlate with the Taxonomy or Phylogeny in Avian Species

PubMed Central

FUJISAWA, Nozomi; KAWAI, Yusuke K.; NAKAYAMA, Shouta M. M.; IKENAKA, Yoshinori; YAMAMOTO, Hideaki; ISHIZUKA, Mayumi

2013-01-01

ABSTRACT There are two arylhydrocarbon receptor (AhR) isoforms in birds, AhR1 and AhR2. The varying sensitivity of AhR is reported to be related to two critical amino acids at positions 325 and 381 in the AhR1 ligand-binding domain. In this study, seven avian species whose in vivo dioxin sensitivity was known, and 13 species with no data regarding their in vivo dioxin sensitivity were examined. The two critical amino acids in the ligand-binding domain were investigated in avian species, and the results were compared with the taxonomy or phylogenetic trees for the bird AhR proteins. We found that the two critical amino acids did not correlate with the taxonomy or phylogeny of these proteins, suggesting that dioxin sensitivity was independent of taxonomy. PMID:23912877

Multivalent Binding and Biomimetic Cell Rolling Improves the Sensitivity and Specificity of Circulating Tumor Cell Capture.

PubMed

Myung, Ja Hye; Eblan, Michael J; Caster, Joseph M; Park, Sin-Jung; Poellmann, Michael J; Wang, Kyle; Tam, Kevin A; Miller, Seth M; Shen, Colette; Chen, Ronald C; Zhang, Tian; Tepper, Joel E; Chera, Bhishamjit S; Wang, Andrew Z; Hong, Seungpyo

2018-06-01

Purpose: We aimed to examine the effects of multivalent binding and biomimetic cell rolling on the sensitivity and specificity of circulating tumor cell (CTC) capture. We also investigated the clinical significance of CTCs and their kinetic profiles in patients with cancer undergoing radiotherapy treatment. Experimental Design: Patients with histologically confirmed primary carcinoma undergoing radiotherapy, with or without chemotherapy, were eligible for enrollment. Peripheral blood was collected prospectively at up to five time points, including before radiotherapy, at the first week, mid-point and final week of treatment, as well as 4 to 12 weeks after completion of radiotherapy. CTC capture was accomplished using a nanotechnology-based assay (CapioCyte) functionalized with aEpCAM, aHER-2, and aEGFR. Results: CapioCyte was able to detect CTCs in all 24 cancer patients enrolled. Multivalent binding via poly(amidoamine) dendrimers further improved capture sensitivity. We also showed that cell rolling effect can improve CTC capture specificity (% of captured cells that are CK + /CD45 - /DAPI + ) up to 38%. Among the 18 patients with sequential CTC measurements, the median CTC decreased from 113 CTCs/mL before radiotherapy to 32 CTCs/mL at completion of radiotherapy ( P = 0.001). CTCs declined throughout radiotherapy in patients with complete clinical and/or radiographic response, in contrast with an elevation in CTCs at mid or post-radiotherapy in the two patients with known pathologic residual disease. Conclusions: Our study demonstrated that multivalent binding and cell rolling can improve the sensitivity and specificity of CTC capture compared with multivalent binding alone, allowing reliable monitoring of CTC changes during and after treatment. Clin Cancer Res; 24(11); 2539-47. ©2018 AACR . ©2018 American Association for Cancer Research.

An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

PubMed

Holland, Erika B; Feng, Wei; Zheng, Jing; Dong, Yao; Li, Xueshu; Lehmler, Hans-Joachim; Pessah, Isaac N

2017-01-01

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca 2+  channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [ 3 H]Ryanodine ([ 3 H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters.

PubMed

Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y Eugene

2015-10-15

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Proteins with similar architecture exhibit similar large-scale dynamic behavior.

PubMed Central

Keskin, O; Jernigan, R L; Bahar, I

2000-01-01

We have investigated the similarities and differences in the computed dynamic fluctuations exhibited by six members of a protein fold family with a coarse-grained Gaussian network model. Specifically, we consider the cofactor binding fragment of CysB; the lysine/arginine/ornithine-binding protein (LAO); the enzyme porphobilinogen deaminase (PBGD); the ribose-binding protein (RBP); the N-terminal lobe of ovotransferrin in apo-form (apo-OVOT); and the leucine/isoleucine/valine-binding protein (LIVBP). All have domains that resemble a Rossmann fold, but there are also some significant differences. Results indicate that similar global dynamic behavior is preserved for the members of a fold family, and that differences usually occur in regions only where specific function is localized. The present work is a computational demonstration that the scaffold of a protein fold may be utilized for diverse purposes. LAO requires a bound ligand before it conforms to the large-scale fluctuation behavior of the three other members of the family, CysB, PBGD, and RBP, all of which contain a substrate (cofactor) at the active site cleft. The dynamics of the ligand-free enzymes LIVBP and apo-OVOT, on the other hand, concur with that of unliganded LAO. The present results suggest that it is possible to construct structure alignments based on dynamic fluctuation behavior. PMID:10733987

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.

PubMed

Even-Chen, S; Barenholz, Y

2000-12-20

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.

The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide

NASA Astrophysics Data System (ADS)

Żurawska-Płaksej, Ewa; Rorbach-Dolata, Anna; Wiglusz, Katarzyna; Piwowar, Agnieszka

2018-01-01

Albumin, the major serum protein, plays a variety of functions, including binding and transporting endogenous and exogenous ligands. Its molecular structure is sensitive to different environmental modifiers, among which glucose is one of the most significant. In vivo albumin glycation occurs under physiological conditions, but it is increased in diabetes. Since bovine serum albumin (BSA) may serve as a model protein in in vitro experiments, we aimed to investigate the impact of glucose-mediated BSA glycation on the binding capacity towards gliclazide, as well as the ability of this drug to prevent glycation of the BSA molecule. To reflect normo- and hyperglycemia, the conditions of the glycation process were established. Structural changes of albumin after interaction with gliclazide (0-14 μM) were determined using fluorescence quenching and circular dichroism spectroscopy. Moreover, thermodynamic parameters as well as energy transfer parameters were determined. Calculated Stern-Volmer quenching constants, as well as binding constants for the BSA-gliclazide complex, were lower for the glycated form of albumin than for the unmodified protein. The largest, over 2-fold, decrease in values of binding parameters was observed for the sample with 30 mM of glucose, reflecting the poorly controlled diabetic state, which indicates that the degree of glycation had a critical influence on binding with gliclazide. In contrast to significant changes in the tertiary structure of BSA upon binding with gliclazide, only slight changes in the secondary structure were observed, which was reflected by about a 3% decrease of the α-helix content of glycated BSA (regardless of glucose concentration) in comparison to unmodified BSA. The presence of gliclazide during glycation did not affect its progress. The results of this study indicate that glycation significantly changed the binding ability of BSA towards gliclazide and the scale of these changes depended on glucose concentration. It may have a direct impact on the free drug fraction and its pharmacokinetic behavior, including the risk of hypoglycemic episodes or unexpected interactions with other ligands. The use of BSA in examining binding effects upon glycation seems to be good model for preliminary research and may be used to identify a potential drug response in a diabetic state.

Distributed feature binding in the auditory modality: experimental evidence toward reconciliation of opposing views on the basis of mismatch negativity and behavioral measures.

PubMed

Chernyshev, Boris V; Bryzgalov, Dmitri V; Lazarev, Ivan E; Chernysheva, Elena G

2016-08-03

Current understanding of feature binding remains controversial. Studies involving mismatch negativity (MMN) measurement show a low level of binding, whereas behavioral experiments suggest a higher level. We examined the possibility that the two levels of feature binding coexist and may be shown within one experiment. The electroencephalogram was recorded while participants were engaged in an auditory two-alternative choice task, which was a combination of the oddball and the condensation tasks. Two types of deviant target stimuli were used - complex stimuli, which required feature conjunction to be identified, and simple stimuli, which differed from standard stimuli in a single feature. Two behavioral outcomes - correct responses and errors - were analyzed separately. Responses to complex stimuli were slower and less accurate than responses to simple stimuli. MMN was prominent and its amplitude was similar for both simple and complex stimuli, whereas the respective stimuli differed from standards in a single feature or two features respectively. Errors in response only to complex stimuli were associated with decreased MMN amplitude. P300 amplitude was greater for complex stimuli than for simple stimuli. Our data are compatible with the explanation that feature binding in auditory modality depends on two concurrent levels of processing. We speculate that the earlier level related to MMN generation is an essential and critical stage. Yet, a later analysis is also carried out, affecting P300 amplitude and response time. The current findings provide resolution to conflicting views on the nature of feature binding and show that feature binding is a distributed multilevel process.

( sup 3 H)RO15-4513 binding to cerebellar diazepam-sensitive and insensitive GABAA receptors is unchanged by one week of ethanol intake

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, M.W.; Chen, J.P.; Wallis, C.

1992-02-26

({sup 3}H)RO15-4513, a partial inverse agonist at GABAA receptors, binds to two sites in cerebellar membranes, one sensitive (DZ-S) and one insensitive (DZ-IS) to inhibition by diazepam. These binding sites may represent different isoforms of the GABAA receptor and may play a role in ethanol (EtOH) dependence. The authors tested the hypothesis that chronic intake of EtOH induces changes in the binding properties of one or both of these putative GABBA receptors. Rats were fed a liquid diet of 4.5% EtOH for 7 d, gavaged with a 3g/kg dose of EtOH, and then sacrificed after 2 h, 12 h, ormore » 4.5 d. Binding of ({sup 3}H)RO15-4513 to cerebellar membranes was performed in the absence or presence of 10{mu}M diazepam (DZ-IS binding); DZ-S binding was calculated as the difference between total and DZ-IS. Nonlinear regression analysis showed that each class of binding site fit a model of mass action binding to a single, noninteractive population of sites. No significant difference was observed between any of the treatment groups in the apparent affinity (Kd) for ({sup 3}H)RO15-4513 at total, DZ-S, or DZ-IS sites following chronic EtOH intake or withdrawal. In addition, no significant difference was observed in the apparent number of DZ-S or DZ-IS binding sites or the ratio of DZ-S to DZ-IS.« less

Highly Sensitive and Reusable Membraneless Field-Effect Transistor (FET)-Type Tungsten Diselenide (WSe2) Biosensors.

PubMed

Lee, Hae Won; Kang, Dong-Ho; Cho, Jeong Ho; Lee, Sungjoo; Jun, Dong-Hwan; Park, Jin-Hong

2018-05-30

In recent years when the demand for high-performance biosensors has been aroused, a field-effect transistor (FET)-type biosensor (BioFET) has attracted great interest because of its high sensitivity, label-free detection, fast detection speed, and miniaturization. However, the insulating membrane in the conventional BioFET, which is essential in preventing the surface dangling bonds of typical semiconductors from nonspecific bindings, has limited the sensitivity of biosensors. Here, we present a highly sensitive and reusable membraneless BioFET based on a defect-free van der Waals material, tungsten diselenide (WSe 2 ). We intentionally generated a few surface defects that serve as extra binding sites for the bioreceptor immobilization through weak oxygen plasma treatment, consequently magnifying the sensitivity values to 2.87 × 10 5 A/A for 10 mM glucose. The WSe 2 BioFET also maintained its high sensitivity even after several cycles of rinsing and glucose application were repeated.

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

PubMed

D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

2009-01-01

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

Region-specific associations between sex, social status, and oxytocin receptor density in the brains of eusocial rodents.

PubMed

Mooney, S J; Coen, C W; Holmes, M M; Beery, A K

2015-09-10

Naturally occurring variations in neuropeptide receptor distributions in the brain contribute to numerous mammalian social behaviors. In naked mole-rats, which live in large social groups and exhibit remarkable reproductive skew, colony-related social behaviors vary with reproductive status. Here we examined whether variation in social status is associated with variations in the location and/or density of oxytocin binding in this species. Autoradiography was performed to assess forebrain oxytocin receptor (OTR) densities in breeding and non-breeding naked mole-rats of both sexes. Overall, males exhibited higher OTR binding in the medial amygdala in comparison to females. While there were no main effects of reproductive status in any region, a sex difference in OTR binding in the nucleus accumbens was mediated by status. Specifically, breeding males tended to have more OTR binding than breeding females in the nucleus accumbens, while no sex difference was observed in subordinates. These effects suggest that oxytocin may act in a sex- and region-specific way that corresponds to reproductive status and associated social behaviors. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Anchoring antibodies to membranes using a diphtheria toxin T domain-ZZ fusion protein as a pH sensitive membrane anchor.

PubMed

Nizard, P; Liger, D; Gaillard, C; Gillet, D

1998-08-14

We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

PubMed

Patra, Digambara

2010-01-15

A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding*

PubMed Central

Zhou, Xixi; Cooper, Karen L.; Sun, Xi; Liu, Ke J.; Hudson, Laurie G.

2015-01-01

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation. PMID:26063799

Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival.

PubMed

Siede, W; Friedberg, E C

1992-03-01

In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G1/early S and S/G2 phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G1/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.

Characterization and localization of arginine vasotocin receptors in the brain and kidney of an amphibian

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, S.K.

1987-01-01

Because arginine vasotocin (AVT) activates male sexual behaviors in the rough-skinned newt (Taricha granulosa), quantitative autoradiography with radiolabeled arginine vasopressin (/sup 3/H-AVP) was used to localize and characterize putative AVT receptors in the brain of this amphibian. Binding of /sup 3/H-AVP to sites within the medial pallium was saturable, specific, reversible, of high affinity and low capacity. These binding sites appear to represent authentic central nervous system receptors for AVT. Furthermore, ligand specificity for the binding sites in this amphibian differs from that reported for AVP binding sites in rat brains. Dense concentrations of specific binding sites were located inmore » the olfactory nerve as it entered the olfactory bulb within the medial pallium, dorsal pallium, and amygdala pars lateralis of the telencephalon, and in the tegmental region of the medulla. Concentrations of binding sites differed significantly among various brain regions. A comparison of male and female newts collected during the breeding season revealed no sexual dimorphism. These areas may represent site(s) of action where AVT elicits sexual behaviors in male T. granulosa.« less

GABA/benzodiazepine receptor complex in long-sleep and short-sleep mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marley, R.J.

LS mice are more sensitive to benzodiazepine-induced anesthesia; however, the two lines do not differ in their hypothermic response to flurazepam. SS mice are more resistant to 3-mercaptopropionic acid-induced seizures and more sensitive to the anticonvulsant effects of benzodiazepines. The various correlates of GABA and benzodiazepine actions probably are the results of different mechanisms of action and/or differential regional control. Bicuculline competition for /sup 3/H-GABA binding sites is greater in SS cerebellar tissue and /sup 3/H-flunitrazepam binding is greater in the mid-brain region of LS mice. GABA enhancement of /sup 3/H-flunitrazepma binding is greater in SS mice. Ethanol also enhancesmore » /sup 3/H-flunitrazepam binding and increases the levels of /sup 3/H-flunitrazepam binding above those observed for GABA. Using correlational techniques on data from LS and SS mice and several inbred mouse strains, it was demonstrated that a positive relationship exists between the degree of receptor coupling within the GABA receptor complex and the degree of resistance to seizures.« less

Force fields for describing the solution-phase synthesis of shape-selective metal nanoparticles

NASA Astrophysics Data System (ADS)

Zhou, Ya; Al-Saidi, Wissam; Fichthorn, Kristen

2013-03-01

Polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO) are structure-directing agents that exhibit different performance in the polyol synthesis of Ag nanostructures. The success of these structure-directing agents in selective nanostructure synthesis is often attributed to their selective binding to Ag(100) facets. We use first-principles, density-functional theory (DFT) calculations in a vacuum environment to show that PVP has a stronger preference to bind to Ag(100) than to Ag(111), whereas PEO exhibits much weaker selectivity. To understand the role of solvent in the surface-sensitive binding, we develop classical force fields to describe the interactions of the structure-directing (PVP and PEO) and solvent (ethylene glycol) molecules with various Ag substrates. We parameterize the force fields through force-and-energy matching to DFT results using simulated annealing. We validate the force fields by comparisons to DFT and experimental binding energies. Our force fields reproduce the surface-sensitive binding predicted by DFT calculations. Molecular dynamics simulations based on these force fields can be used to reveal the role of solvent, polymer chain length, and polymer concentration in the selective synthesis of Ag nanostructures.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Genetically designed biosensing systems for high-throughput screening of pharmaceuticals, clinical diagnostics, and environmental monitoring

NASA Astrophysics Data System (ADS)

Wenner, Brett R.; Douglass, Phillip; Shrestha, Suresh; Sharma, Bethel V.; Lai, Siyi; Madou, Marc J.; Daunert, Sylvia

2001-05-01

The genetically-modified binding proteins calmodulin, the phosphate binding protein, the sulfate binding protein, and the galactose/glucose binding protein have been successfully employed as biosensing elements for the detection of phenothiazines, phosphate, sulfate, and glucose, respectively. Mutant proteins containing unique cysteine residues were utilized in the site-specific labeling of environment-sensitive fluorescent probes. Changes in the environment of the probes upon ligand-induced conformational changes of the proteins result in changes in fluorescence intensity.

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fay, S.P.; Domalewski, M.D.; Houghton, T.G.

1994-02-01

Environmentally sensitive molecules have many potential cellular applications. The authors have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphthofluorescein), because in previous studies protonation has been used to explain the quenching when the fluorescinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonatedmore » and unprotonated forms can be examined by multiparameter flow cytometry. The authors found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pK[sub a] of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed. 22 refs., 4 figs.« less

Specific binding of trivalent metal ions to λ-carrageenan.

PubMed

Cao, Yiping; Li, Shugang; Fang, Yapeng; Nishinari, Katsuyoshi; Phillips, Glyn O; Lerbret, Adrien; Assifaoui, Ali

2018-04-01

Carrageenans are a family of sulphated cell wall polysaccharides extracted from seaweeds and are widely used in different industrial sectors. Relative to κ-carrageenan (κ-car) and ι-carrageenan (ι-car), the ionic binding behavior of λ-carrageenan (λ-car) is far less studied. In this work, the interaction and binding behavior between λ-car and metal ions of different valency (Na + , K + , Mg 2+ , Ca 2+ , Fe 2+ , Fe 3+ , Al 3+ , Cr 3+ ) have been investigated. In contrast to the non-specific interaction of the monovalent and divalent cations, specific binding has been identified between λ-car and Fe 3+ /Al 3+ . The specific binding could lead to either precipitation or gelation of λ-car, depending on the way of introducing Fe 3+ /Al 3+ ions. Fe 3+ and Al 3+ exhibit the same binding stoichiometry of [M 3+ ]/[repeating unit] = 1.0, with the former having a relatively larger binding constant. Cr 3+ , though having very similar physical properties with Fe 3+ /Al 3+ , is incapable of binding specifically to Cr 3+ . The phenomena could not be interpreted in terms of counterion condensation, and are rather attributable to a mechanism in which hexa-coordination of Fe 3+ /Al 3+ and entropy-driven cation dehydration play crucial roles in driving the binding of the trivalent metal ions to λ-car. Copyright © 2017 Elsevier B.V. All rights reserved.

Tolerance to LSD and DOB induced shaking behaviour: differential adaptations of frontocortical 5-HT(2A) and glutamate receptor binding sites.

PubMed

Buchborn, Tobias; Schröder, Helmut; Dieterich, Daniela C; Grecksch, Gisela; Höllt, Volker

2015-03-15

Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25 mg/kg, i.p.) induce a ketanserin-sensitive (0.5 mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4 ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive [(3)H]spiroperidol and DOB induced [(35)S]GTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive [(3)H]glutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet [(3)H]glutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged [(3)H]glutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in [(3)H]glutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex. Copyright © 2014 Elsevier B.V. All rights reserved.

The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

PubMed Central

Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

2009-01-01

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

PubMed

Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

2009-05-01

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

Behavioral sensitization and cross-sensitization between methylphenidate amphetamine, and 3-4, methylenedioxymethamphetamine (MDMA) in female SD rats

PubMed Central

Yang, Pamela B.; Atkins, Kristal D.; Dafny, Nachum

2014-01-01

The psychostimulants amphetamine and methylphenidate (MPD / Ritalin) are the drugs most often used to treat attention deficit hyperactivity disorder (ADHD). In addition, students of all ages take these drugs to improve academic performance but also abuse them for pleasurable enhancement. In addition, other psychostimulants such 3,4 methylenedioxymethamphetamine (MDMA / ecstasy) are used / abused for similar objectives. One of the experimental markers for the potential of a drug to produce dependence is its ability to induce behavioral sensitization and cross sensitization with other drugs of abuse. The objective of this study is to use identical experimental protocols and behavioral assays to compare in female rats the effects of amphetamine, MPD and MDMA on locomotor activity and to determine if they induce behavioral sensitization and/or cross sensitization with each other. The main findings of this study are 1. Acute amphetamine, MPD and MDMA all elicited increases in locomotor activity. 2. Chronic administration of an intermediate dose of amphetamine or MPD elicited behavioral sensitization. 3. Chronic administration of MDMA elicited behavioral sensitization in some animals and behavioral tolerance in others. 4. Cross sensitization between MPD and amphetamine was observed. 5. MDMA did not show either cross sensitization or cross tolerance with amphetamine. In conclusion, these results suggest that MDMA act by different mechanisms compared to MPD and amphetamine. PMID:21549116

Deconvolution of the role of metal and pH in metal coordinating polymers

NASA Astrophysics Data System (ADS)

Cazzell, Seth; Holten-Andersen, Niels

Nature uses metal binding amino acids to engineer both mechanical properties and structural functionality. Some examples of this metal binding behavior can be found in both mussel foot protein and DNA binding protein. The mussel byssal thread contains reversible intermolecular protein-metal bonds, allowing it to withstand harsh intertidal environments. Zinc fingers form intramolecular protein-metal bonds to stabilize the tertiary structure of DNA binding proteins, allowing specific structural functionality. Inspired by both these metal-binding materials, we present mechanical and spectroscopic characterization of a model polymer system, designed to mimic this bonding. Through these studies, we are able to answer fundamental polymer physics questions, such as the role of pH and metal to ligand ratio, illuminating both the macroscopic and microscopic material behavior. These understandings further bio-inspired engineering techniques that are used to design viscoelastic soft materials. I was supported by the Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program.

Methods of determining complete sensor requirements for autonomous mobility

NASA Technical Reports Server (NTRS)

Curtis, Steven A. (Inventor)

2012-01-01

A method of determining complete sensor requirements for autonomous mobility of an autonomous system includes computing a time variation of each behavior of a set of behaviors of the autonomous system, determining mobility sensitivity to each behavior of the autonomous system, and computing a change in mobility based upon the mobility sensitivity to each behavior and the time variation of each behavior. The method further includes determining the complete sensor requirements of the autonomous system through analysis of the relative magnitude of the change in mobility, the mobility sensitivity to each behavior, and the time variation of each behavior, wherein the relative magnitude of the change in mobility, the mobility sensitivity to each behavior, and the time variation of each behavior are characteristic of the stability of the autonomous system.

Maternal mind-mindedness during infancy, general parenting sensitivity and observed child feeding behavior: a longitudinal study.

PubMed

Farrow, Claire; Blissett, Jackie

2014-01-01

Maternal mind-mindedness, or the tendency to view the child as a mental agent, has been shown to predict sensitive and responsive parenting behavior. As yet the role of mind-mindedness has not been explored in the context of feeding interactions. This study evaluates the relations between maternal mind-mindedness at 6 months of infant age and subsequently observed maternal sensitivity and feeding behaviors with children at age 1 year. Maternal mind-mindedness was greater in mothers who had breast-fed compared to formula-fed. Controlling for breast-feeding, mind-mindedness at 6 months was correlated with observations of more sensitive and positive feeding behaviors at 1 year of age. Mind-mindedness was also associated with greater general maternal sensitivity in play and this general parenting sensitivity mediated the effect of mind-mindedness on more sensitive and positive feeding behaviors. Interventions to promote maternal tendency to consider their child's mental states may encourage more adaptive parental feeding behaviors.

The application of new nanocomposites: Enhancement effect of polylactide nanofibers/nano-TiO 2 blends on biorecognition of anticancer drug daunorubicin

NASA Astrophysics Data System (ADS)

Song, Min; Pan, Chao; Chen, Chen; Li, Jingyuan; Wang, Xuemei; Gu, Zhongze

2008-11-01

In this contribution, the blending of nano-titanium dioxide (TiO 2) and polylactide (PLA) nanofibers has been adopted as a new nanomaterial to facilitate the biorecognition of an anticancer drug daunorubicin. Our observations demonstrate that upon application of the nano-TiO 2-PLA polymer nanocomposites, the drug molecules could be readily self-assembled on the surface of the new nanocomposites so that considerably enhanced detection sensitivity for the DNA binding behavior could be observed for the relative biorecognition. These results may also imply some potential valuable application of the blending of nano-TiO 2 and PLA nanofibers as a kind of drug carriers in view of the respective good biocompatibility and large surface area of the new nanocomposites.

Equilibrium binding behavior of magnesium to wall teichoic acid.

PubMed

Thomas, Kieth J; Rice, Charles V

2015-10-01

Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant. Copyright © 2015 Elsevier B.V. All rights reserved.

ISOLATION OF RABBIT IGA ANTIHAPTEN ANTIBODY AND DEMONSTRATION OF SKIN-SENSITIZING ACTIVITY IN HOMOLOGOUS SKIN

PubMed Central

Onoue, Kaoru; Yagi, Yasuo; Pressman, David

1966-01-01

Multiple antibody components of rabbit antisera against p-azobenzenearsonate (Rp) were studied with respect to their globulin nature and skin-sensitizing activity. IgA antibody was characterized by isolating two IgA-rich fractions from a specifically purified antibody preparation. Examination of these fractions showed that IgA antibodies existed in two molecular forms, one with a sedimentation constant of 7S and the other 9S. Skin-sensitizing activity was examined by a P-K type test and a PCA test with Rp-rabbit serum albumin in homologous (rabbit) species. Only the 7S but not 9S IgA antibody sensitized rabbit skin. IgM antibody showed no activity and IgG antibody showed very low activity. In contrast, only IgG antibody was active in the P-K type test to sensitize a heterologous species (guinea pig). None of the antibodies of other classes showed sensitizing activity in heterologous skin. The 7S IgA antibody lost its sensitizing activity upon reduction and alkylation, although no change in its molecular size could be observed. The loss of sensitizing activity was not due to the destruction of antigen-binding activity since the treated 7S IgA antibody retained this activity as shown by radioimmunoelectrophoresis and by binding to the specific immunoadsorbent. The 9S IgA antibody was more resistant to these treatments than the IgM antibody and showed no indication of dissociation. The treated 9S IgA also retained antigen-binding activity. Both the P-K type and PCA reactions were considerably stronger when the interval between injections of antibody and antigen was 24 hr rather than 4 to 5 hr. PMID:4159250

High levels of wheel running protect against behavioral sensitization to cocaine.

PubMed

Renteria Diaz, Laura; Siontas, Dora; Mendoza, Jose; Arvanitogiannis, Andreas

2013-01-15

Although there is no doubt that the direct action of stimulant drugs on the brain is necessary for sensitization to their behavioral stimulating effects, several experiments indicate that drug action is often not sufficient to produce sensitization. There is considerable evidence that many individual characteristics and experiential variables can modulate the behavioral and neural changes that are seen following repeated exposure to stimulant drugs. In the work presented here, we examined whether chronic wheel running would modulate behavioral sensitization to cocaine, and whether any such influence was contingent on individual differences in wheel running. We found that a 5- or 10-week experience with wheel running protects against behavioral sensitization to cocaine but only in animals with a natural tendency to run the most. Understanding the mechanism underlying the modulating effect of wheel running on behavioral sensitization may have important implications for future studies on the link between drug-induced behavioral and neural adaptations. Copyright © 2012 Elsevier B.V. All rights reserved.

Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice.

PubMed

Haughey, Heather M; Kaiser, Alan L; Johnson, Thomas E; Bennett, Beth; Sikela, James M; Zahniser, Nancy R

2005-10-01

Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

PubMed

Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-11-24

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

PubMed Central

Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

2015-01-01

The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes.

PubMed

Ding, Jiawang; Chen, Yan; Wang, Xuewei; Qin, Wei

2012-02-21

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli

PubMed Central

Landis, Lenore; Xu, Jimin; Johnson, Reid C.

1999-01-01

Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media. Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K+ glutamate with osmoprotecting compounds. This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter. We show that CRP–cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro. Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive. Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity. Osmotic regulation of CRP activity is not limited to proP. Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galΔ4 P1 promoter. These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control. PMID:10601034

The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli.

PubMed

Landis, L; Xu, J; Johnson, R C

1999-12-01

Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media. Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K(+) glutamate with osmoprotecting compounds. This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter. We show that CRP-cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro. Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive. Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity. Osmotic regulation of CRP activity is not limited to proP. Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galdelta4P1 promoter. These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control.

A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.

PubMed

Wiley, J S; Brocklebank, A M; Snook, M B; Jamieson, G P; Sawyer, W H; Craik, J D; Cass, C E; Robins, M J; McAdam, D P; Paterson, A R

1991-02-01

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Resolving distinct molecular origins for copper effects on PAI-1.

PubMed

Bucci, Joel C; McClintock, Carlee S; Chu, Yuzhuo; Ware, Gregory L; McConnell, Kayla D; Emerson, Joseph P; Peterson, Cynthia B

2017-10-01

Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator inhibitor-1 (PAI-1), which is responsible for specific and rapid inhibition of fibrinolytic proteases. Active PAI-1 is inherently unstable and readily converts to a latent, inactive form. The binding of vitronectin and other ligands influences stability of active PAI-1. Our laboratory recently observed reciprocal effects on the stability of active PAI-1 in the presence of transition metals, such as copper, depending on the whether vitronectin was also present (Thompson et al. Protein Sci 20:353-365, 2011). To better understand the molecular basis for these copper effects on PAI-1, we have developed a gel-based copper sensitivity assay that can be used to assess the copper concentrations that accelerate the conversion of active PAI-1 to a latent form. The copper sensitivity of wild-type PAI-1 was compared with variants lacking N-terminal histidine residues hypothesized to be involved in copper binding. In these PAI-1 variants, we observed significant differences in copper sensitivity, and these data were corroborated by latency conversion kinetics and thermodynamics of copper binding by isothermal titration calorimetry. These studies identified a copper-binding site involving histidines at positions 2 and 3 that confers a remarkable stabilization of PAI-1 beyond what is observed with vitronectin alone. A second site, independent from the two histidines, binds metal and increases the rate of the latency conversion.

Theoretical study of strain-dependent optical absorption in a doped self-assembled InAs/InGaAs/GaAs/AlGaAs quantum dot

PubMed Central

Tankasala, Archana; Hsueh, Yuling; Charles, James; Fonseca, Jim; Povolotskyi, Michael; Kim, Jun Oh; Krishna, Sanjay; Allen, Monica S; Allen, Jeffery W; Rahman, Rajib; Klimeck, Gerhard

2018-01-01

A detailed theoretical study of the optical absorption in doped self-assembled quantum dots is presented. A rigorous atomistic strain model as well as a sophisticated 20-band tight-binding model are used to ensure accurate prediction of the single particle states in these devices. We also show that for doped quantum dots, many-particle configuration interaction is also critical to accurately capture the optical transitions of the system. The sophisticated models presented in this work reproduce the experimental results for both undoped and doped quantum dot systems. The effects of alloy mole fraction of the strain controlling layer and quantum dot dimensions are discussed. Increasing the mole fraction of the strain controlling layer leads to a lower energy gap and a larger absorption wavelength. Surprisingly, the absorption wavelength is highly sensitive to the changes in the diameter, but almost insensitive to the changes in dot height. This behavior is explained by a detailed sensitivity analysis of different factors affecting the optical transition energy. PMID:29719758

Modeling hypertrophic IP3 transients in the cardiac myocyte.

PubMed

Cooling, Michael; Hunter, Peter; Crampin, Edmund J

2007-11-15

Cardiac hypertrophy is a known risk factor for heart disease, and at the cellular level is caused by a complex interaction of signal transduction pathways. The IP3-calcineurin pathway plays an important role in stimulating the transcription factor NFAT which binds to DNA cooperatively with other hypertrophic transcription factors. Using available kinetic data, we construct a mathematical model of the IP3 signal production system after stimulation by a hypertrophic alpha-adrenergic agonist (endothelin-1) in the mouse atrial cardiac myocyte. We use a global sensitivity analysis to identify key controlling parameters with respect to the resultant IP3 transient, including the phosphorylation of cell-membrane receptors, the ligand strength and binding kinetics to precoupled (with G(alpha)GDP) receptor, and the kinetics associated with precoupling the receptors. We show that the kinetics associated with the receptor system contribute to the behavior of the system to a great extent, with precoupled receptors driving the response to extracellular ligand. Finally, by reparameterizing for a second hypertrophic alpha-adrenergic agonist, angiotensin-II, we show that differences in key receptor kinetic and membrane density parameters are sufficient to explain different observed IP3 transients in essentially the same pathway.

Control of interference during working memory updating.

PubMed

Szmalec, Arnaud; Verbruggen, Frederick; Vandierendonck, André; Kemps, Eva

2011-02-01

The current study examined the nature of the processes underlying working memory updating. In 4 experiments using the n-back paradigm, the authors demonstrate that continuous updating of items in working memory prevents strong binding of those items to their contexts in working memory, and hence leads to an increased susceptibility to proactive interference. Results of Experiments 1 and 2 show that this interference reflects a competition between a process that reveals the degree of familiarity of an item and a context-sensitive recollection process that depends on the strength of bindings in working memory. Experiment 3 further clarifies the origins of interference during updating by demonstrating that even items that are semantically related to the updated working memory contents but that have not been maintained in working memory before cause proactive interference. Finally, the results of Experiment 4 indicate that the occurrence of interference leads to top-down behavioral adjustments that prioritize recollection over familiarity assessment. The implications of these findings for the construct validity of the n-back task, for the control processes involved in working memory updating, and for the concept of executive control more generally are discussed. (c) 2010 APA, all rights reserved.

The effects of the glycation of transferrin on chromium binding and the transport and distribution of chromium in vivo.

PubMed

Deng, Ge; Dyroff, Samantha L; Lockart, Molly; Bowman, Michael K; Vincent, John B

2016-11-01

Chromium (III) has been shown to act as a pharmacological agent improving insulin sensitivity in rodent models of obesity, insulin resistance, and diabetes. To act in beneficial fashion, chromium must reach insulin-sensitive tissues. Chromium is transported from the bloodstream to the tissues by the iron-transport protein transferrin. When blood concentrations of glucose are high (as in a diabetic subject), transferrin can be glycated, modifying its ability to bind and transport iron. However, the effects of glycation of transferrin on its ability to bind and transport Cr have not been examined previously. Storage of transferrin at 37°C in the presence and absence of glucose has significant effects on the binding of Cr. Transferrin stored in the absence of glucose only binds one equivalent of Cr tightly, compared to the normal binding of two equivalents of Cr by transferrin. Glycated transferrin (stored in the presence of glucose) binds two equivalents of Cr but the changes in its extinction coefficient at 245nm that accompany binding suggest that the Cr-bound transferrin possesses a conformation that deviates appreciably from untreated transferrin. These changes have dramatic effects, greatly reducing the ability of transferrin to transport Cr in vivo in rats. The results suggest that glycation of transferrin in subjects with high blood glucose concentrations should reduce the ability of Cr from pharmacological agents to enter tissues. Copyright © 2016 Elsevier Inc. All rights reserved.

Vig r 6, the cytokinin-specific binding protein from mung bean (Vigna radiata) sprouts, cross-reacts with Bet v 1-related allergens and binds IgE from birch pollen allergic patients’ sera

PubMed Central

Guhsl, Eva Elisabeth; Hofstetter, Gerlinde; Hemmer, Wolfgang; Ebner, Christof; Vieths, Stefan; Vogel, Lothar; Breiteneder, Heimo; Radauer, Christian

2014-01-01

Scope Birch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity. Methods and results Bet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients’ sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients’ sera. Conclusion We demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy. PMID:23996905

Protein adsorption to poly(ethylenimine)-modified Sepharose FF: VI. Partial charge neutralization drastically increases uptake rate.

PubMed

Zhao, Yangyang; Dong, Xiaoyan; Yu, Linling; Sun, Yan

2016-01-04

The adsorption and elution behaviors of bovine serum albumin (BSA) on poly(ethylenimine) (PEI)-grafted Sepharose FF resins were recently studied and a critical ionic capacity (cIC; 600 mmol/L) was found, above which the uptake rate increased drastically due to the occurrence of significant "chain delivery" effect. Moreover, above the cIC value, higher salt concentrations were required for protein elution due to the high charge density of the resins. In this work, we have reduced the charge density on the PEI chains of a PEI-grafted resin by neutralization of the amine groups with sodium acetate. PEI-modified resin with IC of 740 mmol/L (FF-PEI-L740, IC>cIC) was chosen as the starting material, and three resins with residual IC values of 660, 560 and 440 mmol/L (FF-PEI-R440) were obtained. The adsorption and chromatographic behaviors of these resins for BSA were investigated. It was found that, with IC decreasing from 740 to 440 mmol/L, the adsorption capacity kept almost unchanged; the effective protein diffusivity (De) also showed negligible variations as IC decreased from 740 to 560 mmol/L (De/D0=0.38 ± 0.04). However, it was interesting to observe a three-fold increase of the De value for FF-PEI-R440 (De/D0=1.23 ± 0.08). It is considered that the occurrence of the drastic uptake rate increase in FF-PEI-R440 was attributed to the decreased available binding sites for protein molecule, which led to the decrease of binding strength, thus facilitated the happenings of "chain delivery" effect of bound proteins. Besides, a study on the effect of ionic strength clarified that the lower the IC value, the higher the sensitivity of protein binding to salt concentration due to the easily screened electrostatic interactions at low surface charge densities. The ionic strength at the elution peak also decreased with decreasing IC in accordance with the salt sensitivity order. Column breakthrough studies demonstrated that the dynamic adsorption capacity of FF-PEI-R440 was much higher than the other three resins at flow rates higher than 30 cm/h because of its high uptake rate. The findings in this work provided new insights into the effects of the interactions between proteins and grafted polymers on adsorption equilibria and uptake kinetics, which would help the selection and design of suitable media for high-performance protein chromatography. Copyright © 2015. Published by Elsevier B.V.

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations.

PubMed

Lu, Haiting; Huang, Xiaoqin; AbdulHameed, Mohamed Diwan M; Zhan, Chang-Guo

2014-04-01

Molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been performed to explore the dynamic behaviors of cytochrome P450 2A6 (CYP2A6) binding with nicotine analogs (that are typical inhibitors) and to calculate their binding free energies in combination with Poisson-Boltzmann surface area (PBSA) calculations. The combined MD simulations and QM/MM-PBSA calculations reveal that the most important structural parameters affecting the CYP2A6-inhibitor binding affinity are two crucial internuclear distances, that is, the distance between the heme iron atom of CYP2A6 and the coordinating atom of the inhibitor, and the hydrogen-bonding distance between the N297 side chain of CYP2A6 and the pyridine nitrogen of the inhibitor. The combined MD simulations and QM/MM-PBSA calculations have led to dynamic CYP2A6-inhibitor binding structures that are consistent with the observed dynamic behaviors and structural features of CYP2A6-inhibitor binding, and led to the binding free energies that are in good agreement with the experimentally-derived binding free energies. The agreement between the calculated binding free energies and the experimentally-derived binding free energies suggests that the combined MD and QM/MM-PBSA approach may be used as a valuable tool to accurately predict the CYP2A6-inhibitor binding affinities in future computational design of new, potent and selective CYP2A6 inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dextran hydrogel coated surface plasmon resonance imaging (SPRi) sensor for sensitive and label-free detection of small molecule drugs

NASA Astrophysics Data System (ADS)

Li, Shaopeng; Yang, Mo; Zhou, Wenfei; Johnston, Trevor G.; Wang, Rui; Zhu, Jinsong

2015-11-01

The label-free and sensitive detection of small molecule drugs on SPRi is still a challenging task, mainly due to the limited surface immobilization capacity of the sensor. In this research, a dextran hydrogel-coated gold sensor chip for SPRi was successfully fabricated via photo-cross-linking for enhanced surface immobilization capacity. The density of the dextran hydrogel was optimized for protein immobilization and sensitive small molecule detection. The protein immobilization capacity of the hydrogel was 10 times greater than a bare gold surface, and 20 times greater than an 11-mercaptoundecanoic acid (MUA) surface. Such a drastic improvement in immobilization capacity allowed the SPRi sensor to detect adequate response signals when probing small molecule binding events. The binding signal of 4 nM liquid-phase biotin to streptavidin immobilized on the dextran surface reached 435 RU, while no response was observed on bare gold or MUA surfaces. The dextran hydrogel-coated SPRi sensor was also applied in a kinetic study of the binding between an immunosuppressive drug (FK506) and its target protein (FKBP12) in a high-throughput microarray format. The measured binding affinity was shown to be consistent with reported literature values, and a detection limit of 0.5 nM was achieved.

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Niladri; Department of Natural Resource Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, H9X 3V9; Stamler, Christopher J.

2005-05-15

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse,more » mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.« less

The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

PubMed

Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

2009-11-13

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

PubMed

Linden, Anni-Maija; Aller, M Isabel; Leppä, Elli; Rosenberg, Per H; Wisden, William; Korpi, Esa R

2008-10-01

TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

Prenatal ethanol exposure decreases hippocampal /sup 3/H-vinylidene kainic acid binding in 45-day-old rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farr, K.L.; Montano, C.Y.; Paxton, L.L.

1988-11-01

The effect of prenatal ethanol exposure on the kainate-sensitive subtype of glutamate receptor binding sites was studied using in vitro /sup 3/H-vinylidene kainic acid (VKA) autoradiography. Pregnant Sprague-Dawley rats were fed a liquid diet containing either 3.35% or 6.7% ethanol throughout gestation. Pair-fed dams received isocalorically matched liquid diets and a lab chow ad lib group served as control for paired feeding. At 45 days of age, the offspring were sacrificed and their brains analyzed for specific /sup 3/H-VKA binding. Compared to pair-fed controls, specific /sup 3/H-VKA binding was reduced by 13% to 32% in dorsal and ventral hippocampal CA3more » stratum lucidum, entorhinal cortex and cerebellum of 45-day-old rats whose mothers consumed either 3.35% or 6.7% ethanol diets. The binding site reductions were statistically significant only in the ventral hippocampal formation and entorhinal cortex of the 3.35% ethanol diet group rats. Saturation of binding studies in the ventral hippocampal formation of 3.35% ethanol rats indicated that the decrease in specific /sup 3/H-VKA binding was due to a decrease in the total number of binding sites. Given the excitatory effect of kainic acid on the spontaneous firing rate of hippocampal CA3 pyramidal neurons, the reduction of kainate-sensitive glutamate binding in this region is consistent with the electrophysiological observation of decreased spontaneous activity of CA3 pyramidal neurons in fetal alcohol rats.« less

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe.

PubMed

Shvadchak, Volodymyr V; Falomir-Lockhart, Lisandro J; Yushchenko, Dmytro A; Jovin, Thomas M

2011-04-15

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators.

PubMed

Ginsberg, J; Shewring, G; Howells, R; Smith, B R; Hall, R

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Lysergic acid diethylamide (LSD) administration selectively downregulates serotonin2 receptors in rat brain.

PubMed

Buckholtz, N S; Zhou, D F; Freedman, D X; Potter, W Z

1990-04-01

A dosage regimen of lysergic acid diethylamide (LSD) that reliably produces behavioral tolerance in rats was evaluated for effects on neurotransmitter receptor binding in rat brain using a variety of radioligands selective for amine receptor subtypes. Daily administration of LSD [130 micrograms/kg (0.27 mumol/kg) intraperitoneally (IP)] for 5 days produced a decrease in serotonin2 (5-hydroxytryptamine2, 5-HT2) binding in cortex (measured 24 hours after the last drug administration) but did not affect binding to other receptor systems (5-HT1A, 5-HT1B, beta-adrenergic, alpha 1- or alpha 2-adrenergic, D2-dopaminergic) or to a recognition site for 5-HT uptake. The decrease was evident within 3 days of LSD administration but was not demonstrable after the first LSD dose. Following 5 days of LSD administration the decrease was still present 48 hours, but not 96 hours, after the last administration. The indole hallucinogen psilocybin [1.0 mg/kg (3.5 mumol/kg) for 8 days] also produced a significant decrease in 5HT2 binding, but neither the nonhallucinogenic analog bromo-LSD [1.3 mg/kg (2.4 mumol/kg) for 5 days] nor mescaline [10 mg/kg (40.3 mumol/kg) for 5 or 10 days] affected 5-HT2 binding. These observations suggest that LSD and other indole hallucinogens may act as 5-HT2 agonists at postsynaptic 5-HT2 receptors. Decreased 5-HT2 binding strikingly parallels the development and loss of behavioral tolerance seen with repeated LSD administration, but the decreased binding per se cannot explain the gamut of behavioral tolerance and cross-tolerance phenomena among the indole and phenylethylamine hallucinogens.

Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides.

PubMed

de Bruin, Donny; Bossert, Nelli; Aartsma-Rus, Annemieke; Bouwmeester, Dirk

2018-04-06

Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson-Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers

PubMed Central

Chipinda, Itai; Mbiya, Wilbes; Adigun, Risikat Ajibola; Morakinyo, Moshood K.; Law, Brandon F.; Simoyi, Reuben H.; Siegel, Paul D.

2015-01-01

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10−6 M−1 s−1 for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R2 = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers. PMID:24333919

Dynamics simulations for engineering macromolecular interactions

PubMed Central

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-01-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions. PMID:23822508

Behavioral metabolomics analysis identifies novel neurochemical signatures in methamphetamine sensitization

PubMed Central

Adkins, Daniel E.; McClay, Joseph L.; Vunck, Sarah A.; Batman, Angela M.; Vann, Robert E.; Clark, Shaunna L.; Souza, Renan P.; Crowley, James J.; Sullivan, Patrick F.; van den Oord, Edwin J.C.G.; Beardsley, Patrick M.

2014-01-01

Behavioral sensitization has been widely studied in animal models and is theorized to reflect neural modifications associated with human psychostimulant addiction. While the mesolimbic dopaminergic pathway is known to play a role, the neurochemical mechanisms underlying behavioral sensitization remain incompletely understood. In the present study, we conducted the first metabolomics analysis to globally characterize neurochemical differences associated with behavioral sensitization. Methamphetamine-induced sensitization measures were generated by statistically modeling longitudinal activity data for eight inbred strains of mice. Subsequent to behavioral testing, nontargeted liquid and gas chromatography-mass spectrometry profiling was performed on 48 brain samples, yielding 301 metabolite levels per sample after quality control. Association testing between metabolite levels and three primary dimensions of behavioral sensitization (total distance, stereotypy and margin time) showed four robust, significant associations at a stringent metabolome-wide significance threshold (false discovery rate < 0.05). Results implicated homocarnosine, a dipeptide of GABA and histidine, in total distance sensitization, GABA metabolite 4-guanidinobutanoate and pantothenate in stereotypy sensitization, and myo-inositol in margin time sensitization. Secondary analyses indicated that these associations were independent of concurrent methamphetamine levels and, with the exception of the myo-inositol association, suggest a mechanism whereby strain-based genetic variation produces specific baseline neurochemical differences that substantially influence the magnitude of MA-induced sensitization. These findings demonstrate the utility of mouse metabolomics for identifying novel biomarkers, and developing more comprehensive neurochemical models, of psychostimulant sensitization. PMID:24034544

Localization of serotoni (5-hydroxytryptamine, 5-HT) with partial purification and characterization of a serotonin binding protein in the intestinal tissue of the nematode Ascaris suum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, R.E.

1989-01-01

An intracellular 5-HT binding protein (SBP) from intestinal tissue was partially purified and characterized. Binding of ({sup 3}H) 5-HT to the protein appeared to be Fe{sup +2}-sensitive and maximal (20.8pmol/mg protein) at 5 {times} 10{sup {minus}4}M Fe{sup +2} and 10{sup {minus}7}M ({sup 3}H) 5-HT. There were two 5-HT binding sites present at optimum Fe{sup +2} concentrations. The Bmax values of these sites were more sensitive to Fe{sup +2} than Kd values. Sulfhydryl reducing agents, cation chelators, Fe{sup +3}, Ca{sup +2} and antagonists of 5-HT uptake and storage inhibited binding of 5-HT to SBP. Gel exclusion chromatography indicated the presence ofmore » a 45Kda SBP that in 5 {times} 10{sup {minus}5}M Fe{sup +2} may form aggregates ranging in size from approximately 80 to >1000Kda. The data indicate these in vitro aggregates may correspond to the electron-opaque patches observed in situ. Ascaris suum may provide a model system to further elucidate the physiological role of analogous serotonin binding proteins that have been identified in mammalian systems.« less

Aptamer-based SERRS Sensor for Thrombin Detection

PubMed Central

Cho, Hansang; Baker, Brian R.; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V.; Laurence, Ted A.; Lane, Stephen M.; Lee, Luke P.; Tok, Jeffrey B.-H.

2012-01-01

We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human α-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes. PMID:19367849

Engineering vanilloid-sensitivity into the rat TRPV2 channel

PubMed Central

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-01-01

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001 PMID:27177419

Engineering vanilloid-sensitivity into the rat TRPV2 channel.

PubMed

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-05-13

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA

PubMed Central

Berke, Ian C; Modis, Yorgo

2012-01-01

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16–18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5–RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry. PMID:22314235

Dopamine agonist suppression of rapid-eye-movement sleep is secondary to sleep suppression mediated via limbic structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miletich, R.S.

The effects of pergolide, a direct dopamine receptor agonist, on sleep and wakefulness, motor behavior and /sup 3/H-spiperone specific binding in limbic structures and striatum in rats was studied. The results show that pergolide induced a biphasic dose effect, with high doses increasing wakefulness and suppressing sleep while low dose decreased wakefulness, but increased sleep. It was shown that pergolide-induced sleep suppression was blocked by ..cap alpha..-glupenthixol and pimozide, two dopamine receptor antagonists. It was further shown that pergolide merely delayed the rebound resulting from rapid-eye-movement (REM) sleep deprivation, that dopamine receptors stimulation had no direct effect on the period,more » phase or amplitude of the circadian rhythm of REM sleep propensity and that there was no alteration in the coupling of REM sleep episodes with S/sub 2/ episodes. Rapid-eye-movement sleep deprivation resulted in increased sensitivity to the pergolide-induced wakefulness stimulation and sleep suppression and pergolide-induced motor behaviors of locomotion and head bobbing. /sup 3/H-spiperone specific binding to dopamine receptors was shown to be altered by REM sleep deprivation in the subcortical limbic structures. It is concluded that the REM sleep suppressing action of dopamine receptor stimulation is secondary to sleep suppression per se and not secondary to a unique effect on the REM sleep. Further, it is suggested that the wakefulness stimulating action of dopamine receptor agonists is mediated by activation of the dopamine receptors in the terminal areas of the mesolimbocortical dopamine projection system.« less

KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins.

PubMed

Zhong, Zhenyu; Pannu, Vaishali; Rosenow, Matthew; Stark, Adam; Spetzler, David

2018-06-04

The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular function is yet to be understood. Here we show that silencing KIAA0100 by siRNA in the breast cancer cell line MDA-MB-231 significantly reduced the cancer cells' aggressive behavior, including cell aggregation, reattachment, cell metastasis and invasion. Most importantly, silencing the expression of KIAA0100 particularly sensitized the quiescent cancer cells in suspension culture to anoikis. Immunoprecipitation, mass spectrometry and immunofluorescence analysis revealed that KIAA0100 may play multiple roles in the cancer cells, including stabilizing microtubule structure as a microtubule binding protein, and contributing to MDA-MB-231 cells Anoikis resistance by the interaction with stress protein HSPA1A. Our study also implies that the interaction between KIAA0100 and HSPA1A may be targeted for new drug development to specifically induce anoikis cell death in the cancer cell.

Characterization of Two Mutations, M287L and Q266I, in the α1 Glycine Receptor Subunit That Modify Sensitivity to Alcohols

PubMed Central

Borghese, Cecilia M.; Blednov, Yuri A.; Quan, Yu; Iyer, Sangeetha V.; Xiong, Wei; Mihic, S. John; Zhang, Li; Lovinger, David M.; Trudell, James R.; Homanics, Gregg E.

2012-01-01

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317–329, 2012). PMID:22037201

Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ooko, Edna

Background: Multidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methods: Cytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC{sub 50} values and binding energies. Results: The compounds displayed IC{sub 50} values between 0.7more » ± 0.03 and 20.2 ± 0.25 μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from − 9.00 ± 0.10 to − 6.20 ± 0.02 kcal/mol and pKi values from 0.24 ± 0.04 to 29.17 ± 0.88 μM. At the ATP-binding site of P-gp, lowest binding energies ranged from − 9.78 ± 0.17 to − 6.79 ± 0.01 kcal/mol and pKi values from 0.07 ± 0.02 to 0.03 ± 0.03 μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R = 0.797 and R = 0.794 for training and test sets). Conclusion: Selected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR. - Highlights: • Novel derivatives of curcumin in reversing multidrug resistance (MDR) • Biological and Insilco assays to assess effect on P-glycoprotein (P-gp) • Curcumin synthetic derivatives as possible lead compound against multidrug resistant cancer.« less

Characteristics of ethanol-induced behavioral sensitization in rats: Molecular mediators and cross-sensitization between ethanol and cocaine.

PubMed

Xu, Shijie; Kang, Ung Gu

2017-09-01

Repeated exposure to drugs of abuse can induce a progressive increase in locomotor activity, known as behavioral sensitization. However, little is known about behavioral sensitization to ethanol. We examined whether ethanol could induce behavioral sensitization and investigated several molecular changes accompanying sensitization. We also assessed whether "cross-sensitization" occurred between ethanol and cocaine, another abused drug. Ethanol-induced sensitization was examined in rats after ethanol treatment (0.5 or 2g/kg) for 15days. The biochemical effects of low- or high-dose ethanol were examined in terms of N-methyl-d-aspartate (NMDA) receptor subunit phosphorylation or expression. Neuronal activity after ethanol treatment was assessed by measuring the level of early growth response (Egr-1) expression. Ethanol-induced behavioral sensitization was observed at the low dose (0.5g/kg) but not the high dose (2g/kg). Although acute treatment with the sensitizing dose of ethanol robustly increased Egr-1 protein and mRNA levels, the expression and phosphorylation of NMDA receptor subunits were not affected. The biochemical responses to ethanol seemed to be enhanced in ethanol-sensitized animals. Cross-sensitization between ethanol and cocaine was observed, which supports the hypothesis that there are commonalities among substances in the pathophysiology of substance dependence. Copyright © 2017 Elsevier Inc. All rights reserved.

Temporal concordance of anorectic, behavioral, cardiovascular and amphetamine receptor binding activity of phenethylamines in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borrelli, A.; Blosser, J.; Barrantes, M.

Although numerous studies have described the anorectic, cardiovascular, and behavioral effects of phenthylamines, a comparison of the pharmacological concordance of these properties in a single species is needed. The objectives of this study were to compare the anorectic potency of 13 phenethylamines following po administration with their effects on spontaneous locomotor activity (SLA) and blood pressure (BP) in vivo and with amphetamine receptor affinity in vitro. The anorectic potencies (ED 50) ranged from 12 umol/kg (fenfluramine) to over 400 umol/kg (d-norephedrine and 1-pseudoephedrine). d-Amphetamine, phentermine, and d-norpseudoephedrine were among the most active and 1-pseudoephedrine and 1-nor-ephedrine the least active inmore » increasing SLA. 1-Norephedrine, and d-norpseudoephedrine were the most active increasing BP while d-norephedrine produced a weak vasodepressor effect. A significant correlation (r = .80) was observed between anorectic potency and affinity (IC 50) for /sup 3/H-amphetamine binding sites in the hypothalamus. However, the stereoselectivity between pairs of enantiomers to inhibit food consumption was not paralleled in binding affinity. The rank order of concordance of phenethylamines in anorectic activity was most apparent in behavior and binding affinity.« less

[Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Binding of L-[3H]glutamate to synaptic membranes of the rat cerebral cortex].

PubMed

Dambinova, S A; Gorodinskiĭ, A I

1984-01-01

The binding of L-[3H]glutamate to rat cerebral cortex synaptic membranes was investigated. Two types of binding sites, a Na+-independent (Kd = 140-160 nm; Bmax = 3.8-4.5 pmol-mg of protein) and a Na+-dependent (Kd = 2.0 microM; Bmax = 45-50 pmol/mg of protein) ones, were detected. The dependence of Na+-insensitive binding on time and temperature and membrane content in a sample was determined. Mono- and divalent cations (5-10 mM) potentiated specific binding by 2.1-3.3 times. The Na+-dependent binding is associated with active transport systems, while the Na+-independent one-with true receptor binding. The relationship between CNS glutamate receptors and Na+-independent binding sites is discussed.

Effects of weak/non-complement-binding HLA antibodies on C1q-binding.

PubMed

Hönger, G; Amico, P; Arnold, M-L; Spriewald, B M; Schaub, S

2017-08-01

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Importance of associative learning processes for one-trial behavioral sensitization of preweanling rats.

PubMed

McDougall, Sanders A; Pothier, Alexandria G; Der-Ghazarian, Taleen; Herbert, Matthew S; Kozanian, Olga O; Castellanos, Kevin A; Flores, Ana T

2011-10-01

During adulthood, associative learning is necessary for the expression of one-trial behavioral sensitization; however, it is uncertain whether the same associative processes are operative during the preweanling period. Two strategies were used to assess the importance of associative learning for one-trial behavioral sensitization of preweanling rats. In the initial experiments, we varied both the sequence and time interval between presentation of the conditioned stimulus (CS, novel environment) and unconditioned stimulus (US, cocaine). In the final experiment, we determined whether electroconvulsive shock-induced retrograde amnesia would disrupt one-trial behavioral sensitization. Results showed that robust-sensitized responding was apparent regardless of the sequence in which cocaine and the novel environment (the presumptive CS) were presented. Varying the time between CS and US presentation (0, 3, or 6 h) was also without effect. Results from experiment 3 showed that single or multiple electroconvulsive shock treatments did not alter the expression of the sensitized response. Therefore, these data indicated that one-trial behavioral sensitization of preweanling rats was exclusively mediated by nonassociative mechanisms and that associative processes did not modulate sensitized responding. These findings are in contrast to what is observed during adulthood, as adult rats exhibit one-trial behavioral sensitization only when associative processes are operative.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

PubMed

Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

2001-02-28

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1

PubMed Central

Kim, Kwang Seok; Choi, Kyu Jin; Bae, Sangwoo

2016-01-01

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1-NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1-NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1-NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1-NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1-NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents. PMID:28025997

Classification and virtual screening of androgen receptor antagonists.

PubMed

Li, Jiazhong; Gramatica, Paola

2010-05-24

Computational tools, such as quantitative structure-activity relationship (QSAR), are highly useful as screening support for prioritization of substances of very high concern (SVHC). From the practical point of view, QSAR models should be effective to pick out more active rather than inactive compounds, expressed as sensitivity in classification works. This research investigates the classification of a big data set of endocrine-disrupting chemicals (EDCs)-androgen receptor (AR) antagonists, mainly aiming to improve the external sensitivity and to screen for potential AR binders. The kNN, lazy IB1, and ADTree methods and the consensus approach were used to build different models, which improve the sensitivity on external chemicals from 57.1% (literature) to 76.4%. Additionally, the models' predictive abilities were further validated on a blind collected data set (sensitivity: 85.7%). Then the proposed classifiers were used: (i) to distinguish a set of AR binders into antagonists and agonists; (ii) to screen a combined estrogen receptor binder database to find out possible chemicals that can bind to both AR and ER; and (iii) to virtually screen our in-house environmental chemical database. The in silico screening results suggest: (i) that some compounds can affect the normal endocrine system through a complex mechanism binding both to ER and AR; (ii) new EDCs, which are nonER binders, but can in silico bind to AR, are recognized; and (iii) about 20% of compounds in a big data set of environmental chemicals are predicted as new AR antagonists. The priority should be given to them to experimentally test the binding activities with AR.

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Silencing of Chemosensory Protein Gene NlugCSP8 by RNAi Induces Declining Behavioral Responses of Nilaparvata lugens

PubMed Central

Waris, Muhammad I.; Younas, Aneela; ul Qamar, Muhammad T.; Hao, Liu; Ameen, Asif; Ali, Saqib; Abdelnabby, Hazem Elewa; Zeng, Fang-Fang; Wang, Man-Qun

2018-01-01

Chemosensory proteins (CSPs) play imperative functions in chemical and biochemical signaling of insects, as they distinguish and transfer ecological chemical indications to a sensory system in order to initiate behavioral responses. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), has emerged as the most destructive pest, causing serious damage to rice in extensive areas throughout Asia. Biotic characteristics like monophagy, dual wing forms, and annual long-distance migration imply a critical role of chemoreception in N. lugens. In this study, we cloned the full-length CSP8 gene from N. lugens. Protein sequence analysis indicated that NlugCSP8 shared high sequence resemblance with the CSPs of other insect family members and had the typical four-cysteine signature. Analysis of gene expression indicated that NlugCSP8 mRNA was specifically expressed in the wings of mated 3-day brachypterous females with a 175-fold difference compare to unmated 3-day brachypterous females. The NlugCSP8 mRNA was also highly expressed in the abdomen of unmated 5-day brachypterous males and correlated to the age, gender, adult wing form, and mating status. A competitive ligand-binding assay demonstrated that ligands with long chain carbon atoms, nerolidol, hexanal, and trans-2-hexenal were able to bind to NlugCSP8 in declining order of affinity. By using bioinformatics techniques, three-dimensional protein structure modeling and molecular docking, the binding sites of NlugCSP8 to the volatiles which had high binding affinity were predicted. In addition, behavioral experiments using the compounds displaying the high binding affinity for the NlugCSP8, revealed four compounds able to elicit significant behavioral responses from N. lugens. The in vivo functions of NlugCSP8 were further confirmed through the testing of RNAi and post-RNAi behavioral experiments. The results revealed that reduction in NlugCSP8 transcript abundance caused a decrease in behavioral response to representative attractants. An enhanced understanding of the NlugCSP8 is expected to contribute in the improvement of more effective and eco-friendly control strategies of BPH. PMID:29706901

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

PubMed

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

Lessons learned from participating in D3R 2016 Grand Challenge 2: compounds targeting the farnesoid X receptor

NASA Astrophysics Data System (ADS)

Duan, Rui; Xu, Xianjin; Zou, Xiaoqin

2018-01-01

D3R 2016 Grand Challenge 2 focused on predictions of binding modes and affinities for 102 compounds against the farnesoid X receptor (FXR). In this challenge, two distinct methods, a docking-based method and a template-based method, were employed by our team for the binding mode prediction. For the new template-based method, 3D ligand similarities were calculated for each query compound against the ligands in the co-crystal structures of FXR available in Protein Data Bank. The binding mode was predicted based on the co-crystal protein structure containing the ligand with the best ligand similarity score against the query compound. For the FXR dataset, the template-based method achieved a better performance than the docking-based method on the binding mode prediction. For the binding affinity prediction, an in-house knowledge-based scoring function ITScore2 and MM/PBSA approach were employed. Good performance was achieved for MM/PBSA, whereas the performance of ITScore2 was sensitive to ligand composition, e.g. the percentage of carbon atoms in the compounds. The sensitivity to ligand composition could be a clue for the further improvement of our knowledge-based scoring function.

RNA–protein binding kinetics in an automated microfluidic reactor

PubMed Central

Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

2009-01-01

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214

miR-128 modulates chemosensitivity and invasion of prostate cancer cells through targeting ZEB1.

PubMed

Sun, Xianglun; Li, Youkong; Yu, Jie; Pei, Hong; Luo, Pengcheng; Zhang, Jie

2015-05-01

Recent reports strongly suggest the profound role of miRNAs in cancer therapeutic response and progression, including invasion and metastasis. The sensitivity to therapy and invasion is the major obstacle for successful treatment in prostate cancer. We aimed to investigate the regulative effect of miR-128/zinc-finger E-box-binding homeobox 1 axis on prostate cancer cell chemosensitivity and invasion. The miR-128 expression pattern of prostate cancer cell lines and tissues was detected by real-time reverse transcriptase-polymerase chain reaction, while the mRNA and protein expression levels of zinc-finger E-box-binding homeobox 1 were measured by real-time reverse transcriptase-polymerase chain reaction and western blot assay, respectively. Dual-luciferase reporter gene assay was used to find the direct target of miR-128. Furthermore, prostate cancer cells were treated with miR-128 mimic or zinc-finger E-box-binding homeobox 1-siRNA, and then the cells' chemosensitivity and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and transwell assay, respectively. We found miR-128 expression obviously decreased in prostate cancer tissues compared with paired normal tissues. Restored miR-128 expression sensitized prostate cancer cells to cisplatin and inhibited the invasion. Furthermore, there was an inverse expression pattern between miR-128 and zinc-finger E-box-binding homeobox 1 in prostate cancer cells and tissues, and zinc-finger E-box-binding homeobox 1 was identified as a direct target of miR-128 in prostate cancer. Knockdown of zinc-finger E-box-binding homeobox 1 expression efficiently sensitized prostate cancer cells to cisplatin and inhibited the invasion. However, ectopic zinc-finger E-box-binding homeobox 1 expression impaired the effects of miR-128 on chemosensitivity and invasion in prostate cancer cells. miR-128 functions as a potential cancer suppressor in prostate cancer progression and rational therapeutic strategies for prostate cancer would be developed based on miR-128/zinc-finger E-box-binding homeobox 1 axis. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Early-life stress links 5-hydroxymethylcytosine to anxiety-related behaviors

PubMed Central

Papale, Ligia A.; Madrid, Andy; Li, Sisi; Alisch, Reid S.

2017-01-01

ABSTRACT Environmental stress contributes to the development of psychiatric disorders, including posttraumatic stress disorder and anxiety. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in the brain and is associated with active transcription of neuronal genes. Here we examined behavioral and molecular alterations in adult mice that experienced an early-life stress before weaning (postnatal day 12 to 18) and found anxiety-like behaviors in adult female mice that were accompanied by correlated disruptions of hypothalamic 5hmC and gene expression in 118 genes, revealing potentially functional 5hmC (i.e., gene regulation). These genes are known and potentially novel stress-related targets, including Nr3c2, Nrxn1, Nfia, and Clip1, that have a significant enrichment for neuronal ontological functions, such as neuronal development and differentiation. Sequence motif predictions indicated that 5hmC may regulate gene expression by mediating transcription factor binding and alternative splicing of many of these transcripts. Together, these findings represent a critical step toward understanding the effects of early environment on the neuromolecular mechanisms that underlie the risk to develop anxiety disorders. PMID:28128679

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria

2016-08-15

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP 2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL -) with a distinct second site is required for high PIP 2sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP 2sensitivity, even in the absence of PL -. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP 2(2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domainmore » (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL -binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP 2site and explaining the positive allostery between PL -binding and PIP 2sensitivity.« less

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids.

PubMed

Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria; Heyman, Sarah; Stary-Weinzinger, Anna; Yuan, Peng; Nichols, Colin G

2016-09-01

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(-)) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL(-) Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(-) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL(-) binding and PIP2 sensitivity. © 2016 Lee et al.

Functionally heterogenous ryanodine receptors in avian cerebellum.

PubMed

Sierralta, J; Fill, M; Suárez-Isla, B A

1996-07-19

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum

PubMed Central

Levine, Amir A.; Guan, Zhonghui; Barco, Angel; Xu, Shiqin; Kandel, Eric R.; Schwartz, James H.

2005-01-01

Remodeling chromatin is essential for cAMP-regulated gene expression, necessary not only for development but also for memory storage and other enduring mental states. Histone acetylation and deacetylation mediate long-lasting forms of synaptic plasticity in Aplysia as well as cognition in mice. Here, we show that histone acetylation by the cAMP-response element binding protein (CREB)-binding protein (CBP) mediates sensitivity to cocaine by regulating expression of the fosB gene and its splice variant, ΔfosB, a transcription factor previously implicated in addiction. Using the chromatin immunoprecipitation assay with antibodies against histone H4 or CBP, we find that CBP is recruited to the fosB promoter to acetylate histone H4 in response to acute exposure to cocaine. We show that mutant mice that lack one allele of the CBP gene and have normal levels of fosB expression are less sensitive to chronic (10-day) administration of cocaine than are wild-type mice. This decreased sensitivity is correlated with decreased histone acetylation and results in decreased fosB expression and diminished accumulation of ΔfosB. Thus, CBP, which forms part of the promoter complex with CREB, mediates sensitivity to cocaine by acetylating histones. PMID:16380431

Ricin, ricin agglutinin, and the ricin binding subunit structural comparison by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Brandt, N. N.; Chikishev, A. Yu.; Sotnikov, A. I.; Savochkina, Yu. A.; Agapov, I. I.; Tonevitsky, A. G.

2005-02-01

Raman spectroscopy is used to study conformation-sensitive vibrational bands of the plant toxins ricin and ricin agglutinin and the ricin binding subunit in aqueous solution. The analysis of the Raman data yields the conformational state of the protein molecules differing from that predicted by the X-ray data. The differences and similarities in the conformational state of ricin, ricin agglutinin, and ricin binding subunit are discussed.

Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogt, B.A.; Gabriel, M.; Vogt, L.J.

Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OxO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OxO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early inmore » training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OxO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OxO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OxO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OxO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OxO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated.« less

Re-entrant phase behavior of a concentrated anionic surfactant system with strongly binding counterions.

PubMed

Ghosh, Sajal Kumar; Rathee, Vikram; Krishnaswamy, Rema; Raghunathan, V A; Sood, A K

2009-08-04

The phase behavior of the anionic surfactant sodium dodecyl sulfate (SDS) in the presence of the strongly binding counterion p-toluidine hydrochloride (PTHC) has been examined using small-angle X-ray diffraction and polarizing microscopy. A hexagonal-to-lamellar transition on varying the PTHC to SDS molar ratio (alpha) occurs through a nematic phase of rodlike micelles (Nc) --> isotropic (I) --> nematic of disklike micelles (N(D)) at a fixed surfactant concentration (phi). The lamellar phase is found to coexist with an isotropic phase (I') over a large region of the phase diagram. Deuterium nuclear magnetic resonance investigations of the phase behavior at phi = 0.4 confirm the transition from N(C) to N(D) on varying alpha. The viscoelastic and flow behaviors of the different phases were examined. A decrease in the steady shear viscosity across the different phases with increasing alpha suggests a decrease in the aspect ratio of the micellar aggregates. From the transient shear stress response of the N() and N(D) nematic phases in step shear experiments, they were characterized to be tumbling and flow aligning, respectively. Our studies reveal that by tuning the morphology of the surfactant micelles strongly binding counterions modify the phase behavior and rheological properties of concentrated surfactant solutions.

Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

NASA Astrophysics Data System (ADS)

Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

2008-11-01

Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times smaller than a cancer cell that functions in a drop of fresh blood.

Stimulation of Suicidal Erythrocyte Death by Tafenoquine.

PubMed

Al Mamun Bhuyan, Abdulla; Bissinger, Rosi; Stockinger, Katja; Lang, Florian

2016-01-01

The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the regulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, zVAD sensitive caspases, SB203580 sensitive p38 kinase, staurosporine sensitive protein kinase C as well as D4476 sensitive casein kinase. The present study explored, whether tafenoquine induces eryptosis and aimed to possibly identify cellular mechanisms involved. Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48 hours exposure of human erythrocytes to tafenoquine (500 ng/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. Tafenoquine did not significantly modify ceramide abundance. The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. The effect of tafenoquine on annexin-V-binding was not significantly blunted by zVAD (10 µM), SB203580 (2 µM) or staurosporine (1 µM). The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by D4476 (10 µM). Tafenoquine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry, oxidative stress and possibly activation of casein kinase. © 2016 The Author(s) Published by S. Karger AG, Basel.

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

PubMed Central

Peter, Daniel; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

2015-01-01

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation. PMID:26294658

The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn{sup 2+} binding to this domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Qing; Li, Chuanyong; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

2015-01-02

Highlights: • The α-helical content of the C-terminus is decreased with a pH increase. • The thermostability of the C-terminus is decreased with a pH increase. • Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues within the C-terminal domain. • The binding of Zn{sup 2+} to His{sup 244} residue is an endothermic heat reaction. • The binding of Zn{sup 2+} to His{sup 266} residue is an exothermic heat reaction. - Abstract: The voltage-gated proton channel Hv1 is strongly sensitive to Zn{sup 2+}. The H{sup +} conduction is decreased at a high concentration of Zn{sup 2+} and Hv1 channelmore » closing is slowed by the internal application of Zn{sup 2+}. Although the recent studies demonstrated that Zn{sup 2+} interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues. The thermodynamics signature of Zn{sup 2+} binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn{sup 2+} to His{sup 244} (mutant H266A) and His{sup 266} (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.« less

Vig r 6, the cytokinin-specific binding protein from mung bean (Vigna radiata) sprouts, cross-reacts with Bet v 1-related allergens and binds IgE from birch pollen allergic patients' sera.

PubMed

Guhsl, Eva Elisabeth; Hofstetter, Gerlinde; Hemmer, Wolfgang; Ebner, Christof; Vieths, Stefan; Vogel, Lothar; Breiteneder, Heimo; Radauer, Christian

2014-03-01

Birch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity. Bet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients' sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients' sera. We demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy. © 2013 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

PubMed Central

Wang, Xue; Zhao, Kun; Kirberger, Michael; Wong, Hing; Chen, Guantao; Yang, Jenny J

2010-01-01

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+-binding pockets of holoproteins to identify such pockets, ignoring possible Ca2+-induced conformational change. In this article, we first report our progress in the analysis of Ca2+-induced conformational changes followed by improved prediction of Ca2+-binding sites in the large group of Ca2+-binding proteins that exhibit only localized conformational changes. The MUGSR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUGSR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUGSR was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca2+-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein. PMID:20512971

Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

PubMed

Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

2016-01-01

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses. Copyright © 2016 the American Physiological Society.

IN VITRO EFFECTS OF CHLORPYRIFOS, PARATHION, METHYL PARATHION AND THEIR OXONS ON CARDIAC MUSCARINIC RECEPTOR BINDING IN NEONATAL AND ADULT RATS. (R825811)

EPA Science Inventory

Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular...

The role of GABA-A and mitochondrial diazepam-binding inhibitor receptors on the effects of neurosteroids on food intake in mice.

PubMed

Reddy, D S; Kulkarni, S K

1998-06-01

The present studies were undertaken to investigate the neuroactive steroidal modulation of feeding behavior and possible involvement of gamma-aminobutyric acid type-A (GABA-A) and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) in food-deprived male mice. Allopregnanolone (0.5-2 mg/kg), a neurosteroid, progesterone (1-10 mg/kg), a neurosteroid precursor, and 4'-chlordiazepam (0.25-1 mg/kg), a specific high affinity MDR agonist, produced a dose-dependent hyperphagic effects. In contrast, neurosteroids pregnenolone sulfate (PS) (1-10 mg/kg) and dehydroepiandrosterone sulfate (DHEAS) (1-10 mg/kg) produced a hypophagic effect, in a dose-dependent manner. The allopregnanolone-, progesterone- and 4'-chlordiazepam-induced hyperphagic effect was blocked by picrotoxin (1 mg/kg), a GABA-A chloride channel antagonist, but not by flumazenil (2 mg/kg), a benzodiazepine (BZD) antagonist. The 4'-chlordiazepam-induced hyperphagic effect was prevented by pretreatment with PK11195 (2 mg/kg), a selective partial MDR antagonist. The hypophagic effect of DHEAS (10 mg/kg) was reversed by dizocilpine (10 microg/kg), an NMDA receptor antagonist, but resistant to muscimol (0.1 mg/kg), a selective GABA-A receptor agonist. In contrast, the PS (10 mg/kg)-induced hypophagic response was resistant to dizocilpine, but sensitive to muscimol (0.1 mg/kg). Both the sulfated neurosteroids PS and DHEAS also reversed the hyperphagic effect of allopregnanolone. In addition, the BZD agonist triazolam (0.05-0.25 mg/kg) also produced a flumazenil- and picrotoxin-sensitive hyperphagic effects, thereby suggesting the changes in feeding behavior by neurosteroids represent GABA-A receptor mediated hyperphagic action. Although the possible antistress or anxiolytic actions of neurosteroids may confound the hyperphagia, behavioral effects observed were specific to food because the mice were adopted to the test environment and diet, and of a possible variation between various neurosteroids in the extent to which antistress or anxiolytic effect produced at hyperphagic doses. The hyperphagic effects of progesterone and 4'-chlordiazepam resembled that of neurosteroid allopregnanolone. Therefore, the effect of progesterone may be imputed to its metabolism to allopregnanolone, while the 4'-chlordiazepam-induced hyperphagic response is related to its MDR-stimulated neurosteroidogenesis and subsequent modulation of GABA-A receptors. The hypophagic response following DHEAS may, at least partly, involve an NMDA receptor mechanism. However, PS-induced hypophagia may be mediated by GABA-A or other receptor systems. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the hyperphagic effects of neurosteroids and reinforces a role for endogenous neurosteroids in regulating feeding behavior. Future studies may lead to the development of neurosteroid-based anorectic/hyperphagic agents for therapeutic use.

Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain.

PubMed

Blane, Ashleigh; Fanucchi, Sylvia

2015-06-30

Forkhead box P2 (FOXP2) is a transcription factor expressed in cardiovascular, intestinal, and neural tissues during embryonic development and is implicated in language development. FOXP2 like other FOX proteins contains a DNA binding domain known as the forkhead domain (FHD). The FHD interacts with DNA by inserting helix 3 into the major groove. One of these DNA-protein interactions is a direct hydrogen bond that is formed with His554. FOXP2 is localized in the nuclear compartment that has a pH of 7.5. Histidine contains an imidazole side chain in which the amino group typically has a pKa of ~6.5. It seems possible that pH fluctuations around 6.5 may result in changes in the protonation state of His554 and thus the ability of the FOXP2 FHD to bind DNA. To investigate the effect of pH on the FHD, both the structure and the binding affinity were studied in the pH range of 5-9. This was done in the presence and absence of DNA. The structure was assessed using size exclusion chromatography, far-UV circular dichroism, and intrinsic and extrinsic fluorescence. The results indicated that while pH did not affect the secondary structure in the presence or absence of DNA, the tertiary structure was pH sensitive and the protein was less compact at low pH. Furthermore, the presence of DNA caused the protein to become more compact at low pH and also had the potential to increase the dimerization propensity. Fluorescence anisotropy was used to investigate the effect of pH on the FOXP2 FHD DNA binding affinity. It was found that pH had a direct effect on binding affinity. This was attributed to the altered hydrogen bonding patterns upon protonation or deprotonation of His554. These results could implicate pH as a means of regulating transcription by the FOXP2 FHD, which may also have repercussions for the behavior of this protein in cancer cells.

Membrane-Dependent Effects of a Cytoplasmic Helix on the Structure and Drug Binding of the Influenza Virus M2 Protein

PubMed Central

Cady, Sarah; Wang, Tuo; Hong, Mei

2011-01-01

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22 to 46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45 to 62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21–61). 13C-2H distance measurements of 13C-labeled protein and 2H-labeled amantadine showed that in DMPC bilayers, the first equivalent of drug bound S31 inside the M2(21–61) pore, similar to the behavior of M2TM in DMPC bilayers. The non-specific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21–61) is S31 in the TM pore. Interestingly, when M2(21–61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, while M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state, but did not rescue drug binding to M2(21–61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helices to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

Light-up fluorescent probes utilizing binding behavior of perylenediimide derivatives to a hydrophobic pocket within DNA.

PubMed

Takada, Tadao; Yamaguchi, Kosato; Tsukamoto, Suguru; Nakamura, Mitsunobu; Yamana, Kazushige

2014-08-21

Here we study the binding behavior of perylenediimide () derivatives to a hydrophobic pocket created inside DNA and their photochemical properties capable of designing a light-up fluorescent sensor for short single-stranded DNA or RNA. The perylenediimide derivative with alkoxy groups () suppressing electron transfer quenching was examined. The bound randomly to DNA showed negligible fluorescence due to the aggregation-induced quenching, whereas the bound to the pocket as a monomeric form showed more than 100-fold fluorescence enhancement. Switching the binding states of the corresponded to a change in the fluorescence response for the hybridization event, which allowed us to design a fluorescent sensor of nucleic acids with a nanomolar detection limit.

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.

PubMed

Naidu, Rayapati A; Ingle, Caroline J; Deom, Carl M; Sherwood, John L

2004-02-05

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

PubMed

Jansson, Keith H; Castillo, Deborah G; Morris, Joseph W; Boggs, Mary E; Czymmek, Kirk J; Adams, Elizabeth L; Schramm, Lawrence P; Sikes, Robert A

2014-01-01

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

PubMed

Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G; Denison, Michael S; Hahn, Mark E; Bonati, Laura; Powell, Wade H

2015-06-02

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

ETMB-RBF: discrimination of metal-binding sites in electron transporters based on RBF networks with PSSM profiles and significant amino acid pairs.

PubMed

Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

2013-01-01

Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation-reduction reactions. In these oxidation-reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins.

ETMB-RBF: Discrimination of Metal-Binding Sites in Electron Transporters Based on RBF Networks with PSSM Profiles and Significant Amino Acid Pairs

PubMed Central

Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

2013-01-01

Background Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation–reduction reactions. In these oxidation–reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. Methods We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. Results We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. Conclusions We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins. PMID:23405059

Regulating the Efficacy of Inhibition Through Trafficking of γ-Aminobutyric Acid Type A Receptors.

PubMed

Vien, Thuy N; Moss, Stephen J; Davies, Paul A

2016-11-01

Trafficking of anesthetic-sensitive receptors within the plasma membrane, or from one cellular component to another, occurs continuously. Changes in receptor trafficking have implications in altering anesthetic sensitivity. γ-Aminobutyric acid type A receptors (GABAARs) are anion-permeable ion channels and are the major class of receptor in the adult mammalian central nervous system that mediates inhibition. GABAergic signaling allows for precise synchronized firing of action potentials within brain circuits that is critical for cognition, behavior, and consciousness. This precision depends upon tightly controlled trafficking of GABAARs into the membrane. General anesthetics bind to and allosterically enhance GABAARs by prolonging the open state of the receptor and thereby altering neuronal and brain circuit activity. Subunit composition and GABAAR localization strongly influence anesthetic end points; therefore, changes in GABAAR trafficking could have significant consequences to anesthetic sensitivity. GABAARs are not static membrane structures but are in a constant state of flux between extrasynaptic and synaptic locations and are continually endocytosed and recycled from and to the membrane. Neuronal activity, posttranslational modifications, and some naturally occurring and synthetic compounds can influence the expression and trafficking of GABAARs. In this article, we review GABAARs, their trafficking, and how phosphorylation of GABAAR subunits can influence the surface expression and function of the receptor. Ultimately, alterations of GABAAR trafficking could modify anesthetic end points, both unintentionally through pathologic processes but potentially as a therapeutic target to adjust anesthetic-sensitive GABAARs.

Biosensor-based approach identifies four distinct calmodulin-binding domains in the G protein-coupled estrogen receptor 1.

PubMed

Tran, Quang-Kim; Vermeer, Mark

2014-01-01

The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+)-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s), we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs). Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+)-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+) indicators identified separate Ca(2+) sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+) response, representing two distinct species (SMD1 sp1 and SMD1 sp2) with drastically different Ca(2+) sensitivities. The Ca(2+) sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+)) values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER-dependent signaling at the receptor level through multiple interaction sites. FRET biosensors represent a simple method to identify unknown calmodulin-binding domains in G protein-coupled receptors and to quantitatively assess binding properties.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

PubMed

Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

2003-04-01

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Cell wall reactivity of acidophilic and alkaliphilic bacteria determined by potentiometric titrations and Cd adsorption experiments.

PubMed

Kenney, Janice P L; Fein, Jeremy B

2011-05-15

In this study, we used potentiometric titrations and Cd adsorption experiments to determine the binding capacities of two acidophilic (A. cryptum and A. acidophilum) and two alkaliphilic (B. pseudofirmus and B. circulans) bacterial species in order to determine if any consistent trends could be observed relating bacterial growth environment to proton and Cd binding properties and to compare those binding behaviors to those of neutrophilic bacteria. All of the bacterial species studied exhibited significant proton buffering over the pH range in this study, with the alkaliphiles exhibiting significantly higher acidity constants than the acidophiles as well as the neutrophilic bacterial consortia. The calculated average site concentrations for each of the bacteria in this study are within 2σ experimental error of each other, with the exception of A. cryptum, which has a significantly higher Site 2 concentration than the other species. Despite differing acidity constants between the acidophiles and alkaliphiles, all bacteria except A. cryptum exhibited remarkably similar Cd adsorption behavior to each other, and the observed extent of adsorption was also similar to that predicted from a generalized model derived using neutrophilic bacterial consortia. This study demonstrates that bacteria that grow under extreme conditions exhibit similar proton and metal adsorption behavior to that of previously studied neutrophilic species and that a single set of proton and metal binding constants can be used to model the behavior of bacterial adsorption under a wide range of environmental conditions.

Glucocorticoid receptor ligand binding in monocytic cells using a microplate assay.

PubMed

Jansen, J; Uitdehaag, B; Koper, J W; van Den Berg, T K

1999-01-01

Glucocorticoids have profound effects on macrophage function and are widely used as anti-inflammatory drugs. Glucocorticoids receptor (GR) ligand binding capacity is a major determinant of cellular glucocorticoid sensitivity. The number and affinity of GR can be measured in a whole cell binding assay using (3)H-dexamethasone. Here, we describe a rapid and simple microplate assay for GR measurement using the human promonocytic cell line THP-1. Copyright 2000 S. Karger AG, Basel.

Handheld Chem/Biosensor Using Extreme Conformational Changes in Designed Binding Proteins to Enhance Surface Plasmon Resonance (SPR)

DTIC Science & Technology

2016-04-01

AFCEC-CX-TY-TR-2016-0007 HANDHELD CHEM/ BIOSENSOR USING EXTREME CONFORMATIONAL CHANGES IN DESIGNED BINDING PROTEINS TO ENHANCE SURFACE PLASMON...Include area code) 03/24/2016 Abstract 08/14/2015--03/31/2016 Handheld chem/ biosensor using extreme conformational changes in designed binding...Baltimore, Maryland on 17-21 April 2016. We propose the development of a highly sensitive handheld chem/ biosensor device using a novel class of engineered

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

PubMed Central

Tan, Benedict G.; Vijgenboom, Erik; Worrall, Jonathan A. R.

2014-01-01

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. PMID:24121681

A fractal analysis of protein to DNA binding kinetics using biosensors.

PubMed

Sadana, Ajit

2003-08-01

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.

RIBEYE(B)-domain binds to lipid components of synaptic vesicles in an NAD(H)-dependent, redox-sensitive manner.

PubMed

Schwarz, Karin; Schmitz, Frank

2017-03-20

Synaptic ribbons are needed for fast and continuous exocytosis in ribbon synapses. RIBEYE is a main protein component of synaptic ribbons and is necessary to build the synaptic ribbon. RIBEYE consists of a unique A-domain and a carboxyterminal B-domain, which binds NAD(H). Within the presynaptic terminal, the synaptic ribbons are in physical contact with large numbers of synaptic vesicle (SV)s. How this physical contact between ribbons and synaptic vesicles is established at a molecular level is not well understood. In the present study, we demonstrate that the RIBEYE(B)-domain can directly interact with lipid components of SVs using two different sedimentation assays with liposomes of defined chemical composition. Similar binding results were obtained with a SV-containing membrane fraction. The binding of liposomes to RIBEYE(B) depends upon the presence of a small amount of lysophospholipids present in the liposomes. Interestingly, binding of liposomes to RIBEYE(B) depends on NAD(H) in a redox-sensitive manner. The binding is enhanced by NADH, the reduced form, and is inhibited by NAD + , the oxidized form. Lipid-mediated attachment of vesicles is probably part of a multi-step process that also involves additional, protein-dependent processes. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anthony, B.L.

The influence of halothane and enflurane (0.5-8%) on muscarinic receptor binding in rat atrium was studied using (/sup 3/H) methylscopolamine ((/sup 3/H)MS). Anesthetic-gas mixtures were blown over membrane suspensions for 20 min before and during the binding assays. Halothane and enflurane increased the affinity of cardiac muscarinic receptors for (/sup 3/H)MS by slowing the rate of dissociation. These anesthetics did not affect the affinity of the receptor for carbamylcholine, but significantly reduced the sensitivity of agonist binding to regulation by guanine nucleotides. For example, the fraction of receptors displaying high affinity agonist binding was decreased by a GTP analog frommore » 0.64 to 0.43 in the absence, but only to 0.52 in the presence of 2% halothane. The binding of a radiolabeled agonist, (/sup 3/H)oxotremorine-M, was reduced by 50% by halothane, while its sensitivity to guanine nucleotides was reduced by at least 100 fold. The diminution of the guanine nucleotide effect may reflect a stabilization of the receptor-G proteincomplex due to either a direct action on the receptor complex or to an alteration of the physical state of the membrane. It is also possible that the ability of the G protein to bind guanine nucleotides is adversely affected by anesthetic agents.« less

The socio-behavioral development of children with symptoms of attachment disorder: An observational study of teacher sensitivity in special education.

PubMed

Spilt, Jantine L; Vervoort, Eleonora; Koenen, Anne-Katrien; Bosmans, Guy; Verschueren, Karine

2016-09-01

Children with Reactive Attachment Disorder (RAD) have serious socio-behavioral problems and often rely on socially abnormal, aggressive, and manipulative forms of communication. Little is known, however, about the influence of teachers on the socio-behavioral development of children with symptoms of RAD. This longitudinal study examined the influence of teacher sensitivity on the socio-behavioral development of children with symptoms of RAD across one school year. The sample included 85 Belgian children and 70 teachers from special education schools. In the previous school year, teachers rated Inhibited and Disinhibited RAD symptoms. In the next school year, teacher Sensitivity was observed in interactions with individual children in the first trimester. Teacher-rated Overt aggression, Relational aggression, and Prosocial behavior was assessed in the first, second, and third trimester. We found no effects of Sensitivity on Prosocial behavior. Also, no effects were found for children with Disinhibited RAD symptoms. For children with Inhibited RAD symptoms, increases in Overt and Relational aggression were observed when Sensitivity was low, whereas decreases were observed when Sensitivity was high. The results suggest that teacher sensitivity is associated with the socio-behavioral development of children with Inhibited RAD symptoms but not with the socio-behavioral development of children with Disinhibited RAD symptoms. Children with Reactive Attachment Disorder (RAD) exhibit socio-behavioral problems that hinder their school adjustment. These socio-behavioral problems appear relatively stable and it is not known what influence special education teachers might have on the development of these problems across a school year. This study suggests that teacher sensitivity is associated with changes in the socio-behavioral development of children with Inhibited RAD symptoms. Whereas high sensitivity was associated with improvements, low sensitivity appeared to exaggerate the socio-behavioral problems of these children. As children with Inhibited RAD symptoms have difficulties communicating their needs and wishes in socially adaptive ways, it may not be easy for teachers to understand these children. Teachers may misinterpret a child's behavior and consequently will fail to respond to the child's underlying needs. This may reinforce the child's socio-behavioral problems and increase the child's reliance on egocentric and aggressive means in interactions with others. This study therefore highlights the need to support teachers in interactions with children with Inhibited RAD symptoms in order to help them understand how the children's observable behaviors in the classroom may convey their underlying socio-emotional needs and how they can respond to these needs. Importantly, teacher sensitivity was not associated with the socio-behavioral development of children with Disinhibited RAD symptoms (e.g., indiscriminate friendliness). Consistent with previous research, this study suggests that children with Inhibited RAD symptoms are more susceptible to the quality of the caregiving environment than children with Disinhibited RAD symptoms and extends this finding to the school context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

PubMed Central

Inoue, Isao; Yanai, Kazuhiko; Kitamura, Daisuke; Taniuchi, Ichiro; Kobayashi, Takashi; Niimura, Kaku; Watanabe, Takehiko; Watanabe, Takeshi

1996-01-01

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter. PMID:8917588

An eye tracking investigation of color-location binding in infants' visual short-term memory.

PubMed

Oakes, Lisa M; Baumgartner, Heidi A; Kanjlia, Shipra; Luck, Steven J

2017-01-01

Two experiments examined 8- and 10-month-old infants' ( N = 71) binding of object identity (color) and location information in visual short-term memory (VSTM) using a one-shot change detection task . Building on previous work using the simultaneous streams change detection task, we confirmed that 8- and 10-month-old infants are sensitive to changes in binding between identity and location in VSTM. Further, we demonstrated that infants recognize specifically what changed in these events. Thus, infants' VSTM for binding is robust and can be observed in different procedures and with different stimuli.

Modification of the kinetic parameters of aldolase on binding to the actin-containing filaments of skeletal muscle.

PubMed Central

Walsh, T P; Clarke, F M; Masters, C J

1977-01-01

The kinetic parameters of fructose bisphosphate aldolase (EC 4.1.2.13) were shown to be modified on binding of the enzyme to the actin-containing filaments of skeletal muscle. Although binding to F-actin or F-actin-tropomyosin filaments results in relative minor changes in kinetic properties, binding to F-actin-tropomyosin-troponin filaments produces major alterations in the kinetic parameters, and, in addition, renders them Ca2+-sensitive. These observations may be relevant to an understanding of the function of this enzyme within the muscle fibre. PMID:889571

Specific binding of magnetic nanoparticle probes to platelets in whole blood detected by magnetorelaxometry

NASA Astrophysics Data System (ADS)

Eberbeck, Dietmar; Wiekhorst, Frank; Steinhoff, Uwe; Schwarz, Kay Oliver; Kummrow, Andreas; Kammel, Martin; Neukammer, Jörg; Trahms, Lutz

2009-05-01

The binding of monoclonal antibodies labelled with magnetic nanoparticles to CD61 surface proteins expressed by platelets in whole blood samples was measured by magnetorelaxometry. This technique is sensitive to immobilization of the magnetic labels upon binding. Control experiments with previous saturation of the epitopes on the platelet surfaces demonstrated the specificity of the binding. The kinetics of the antibody antigen reaction is accessible with a temporal resolution of 12 s. The minimal detectable platelet concentration is about 2000 μL -1 (sample volume 150 μL). The proportionality of the magnetic relaxation amplitude to the number of bound labels allows a quantification of the antibody binding capacity.

Person perception and the bounded rationality of social judgment.

PubMed

Wright, J C; Dawson, V L

1988-11-01

In this article, we develop a bounded rationality view of the relation between person perception and social behavior. Two theses of this approach are that behaviors vary in their significance to observers, and that observers pursue bounded rather than global utility in forming personality impressions. Observers are expected to be sensitive to targets' overall behavioral tendencies and to the variability of their behavior across situations, but both sensitivities are bounded, being greater for behaviors that directly affect observers' outcomes. In two investigations involving extensive hourly and 6-s observations, we examined the bounded utility of people's impressions of personality, demonstrating how impression accuracy is linked to the significance of behaviors. Observers were sensitive to the organization of aggressive behaviors, but less sensitive to the organization of withdrawn behaviors, even when the consistency of those behaviors was comparable. The results clarify the relation between people's inferential shortcomings in laboratory paradigms and the bounded utility of person perception in the natural environment.

High fat diet and food restriction differentially modify the behavioral effects of quinpirole and raclopride in rats.

PubMed

Baladi, Michelle G; France, Charles P

2009-05-21

Nutritional status can impact dopamine systems in a manner that might be important to understanding possible common neurobiological mechanisms that mediate abnormal compulsive food (e.g., obesity) and drug taking. Limiting food intake, for example, can increase sensitivity to the behavioral effects of indirect-acting dopamine receptor agonists. Much less is known regarding possible diet-induced changes in sensitivity to direct-acting dopamine receptor drugs. The present study investigated the effects of a high fat diet and of food restriction on sensitivity of rats to the behavioral effects of a direct-acting dopamine receptor agonist and a dopamine receptor antagonist. Free access to high fat chow increased sensitivity to quinpirole-induced yawning without changing sensitivity to raclopride-induced catalepsy or quinpirole-induced hypothermia. Food restriction (10 g/day) decreased sensitivity to quinpirole-induced yawning and raclopride-induced catalepsy without affecting sensitivity to quinpirole-induced hypothermia. Free access to a standard chow restored sensitivity to the behavioral effects of both drugs in rats that were previously food-restricted but not in rats that previously ate a high fat diet. These data confirm that food restriction can decrease sensitivity to behavioral effects of direct-acting dopamine receptor drugs, they provide evidence (i.e., no change in hypothermic effects) indicating that these changes are not due to pharmacokinetic mechanisms, and they provide initial evidence showing enhanced sensitivity to behavioral effects of dopamine receptor drugs in rats eating a high fat diet. These changes in sensitivity of dopamine systems could be relevant to understanding the impact of nutrition on therapeutic and recreational drug use.

High fat diet and food restriction differentially modify the behavioral effects of quinpirole and raclopride in rats

PubMed Central

Baladi, Michelle G; France, Charles P

2009-01-01

Nutritional status can impact dopamine systems in a manner that might be important to understanding possible common neurobiological mechanisms that mediate abnormal compulsive food (e.g., obesity) and drug taking. Limiting food intake, for example, can increase sensitivity to the behavioral effects of indirect-acting dopamine receptor agonists. Much less is known regarding possible diet-induced changes in sensitivity to direct-acting dopamine receptor drugs. The present study investigated the effects of a high fat diet and of food restriction on sensitivity of rats to the behavioral effects of a direct-acting dopamine receptor agonist and a dopamine receptor antagonist. Free access to high fat chow increased sensitivity to quinpirole-induced yawning without changing sensitivity to raclopride-induced catalepsy or quinpirole-induced hypothermia. Food restriction (10 g/day) decreased sensitivity to quinpirole-induced yawning and raclopride-induced catalepsy without affecting sensitivity to quinpirole-induced hypothermia. Free access to a standard chow restored sensitivity to the behavioral effects of both drugs in rats that were previously food-restricted but not in rats that previously ate a high fat diet. These data confirm that food restriction can decrease sensitivity to behavioral effects of direct-acting dopamine receptor drugs, they provide evidence (i.e., no change in hypothermic effects) indicating that these changes are not due to pharmacokinetic mechanisms, and they provide initial evidence showing enhanced sensitivity to behavioral effects of dopamine receptor drugs in rats eating a high fat diet. These changes in sensitivity of dopamine systems could be relevant to understanding the impact of nutrition on therapeutic and recreational drug use. PMID:19327348

NMR Mapping of Protein Conformational Landscapes using Coordinated Behavior of Chemical Shifts upon Ligand Binding

PubMed Central

Cembran, Alessandro; Kim, Jonggul; Gao, Jiali; Veglia, Gianluigi

2014-01-01

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria following linear trajectories of NMR chemical shifts. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues’ response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein’s approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins. PMID:24604024

Food Approach and Food Avoidance in Young Children: Relation with Reward Sensitivity and Punishment Sensitivity

PubMed Central

Vandeweghe, Laura; Vervoort, Leentje; Verbeken, Sandra; Moens, Ellen; Braet, Caroline

2016-01-01

It has recently been suggested that individual differences in Reward Sensitivity and Punishment Sensitivity may determine how children respond to food. These temperamental traits reflect activity in two basic brain systems that respond to rewarding and punishing stimuli, respectively, with approach and avoidance. Via parent-report questionnaires, we investigate the associations of the general motivational temperamental traits Reward Sensitivity and Punishment Sensitivity with Food Approach and Food Avoidance in 98 preschool children. Consistent with the conceptualization of Reward Sensitivity in terms of approach behavior and Punishment Sensitivity in terms of avoidance behavior, Reward Sensitivity was positively related to Food Approach, while Punishment Sensitivity was positively related to Food Avoidance. Future research should integrate these perspectives (i.e., general temperamental traits Reward Sensitivity and Punishment Sensitivity, and Food Approach and Avoidance) to get a better understanding of eating behavior and related body weight. PMID:27445898

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.

PubMed

Papadakos, Grigorios A; Nastri, Horacio; Riggs, Paul; Dupureur, Cynthia M

2007-05-01

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

Sublethal effects of four insecticides on folding and spinning behavior in the rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae).

PubMed

Yang, Yajun; Wang, Caiyun; Xu, Hongxing; Lu, Zhongxian

2018-03-01

The rice leaffolder, Cnaphalocrocis medinalis, is an important rice pest. The sublethal effects of chlorpyrifos, chlorantraniliprole, emamectin benzoate and spinosad were investigated on the folding and spinning behaviors of third- to fifth-instar C. medinalis larvae (L3 - L5) after insecticidal exposure of the second instar. A 25% lethal concentration (LC 25 ) of chlorpyrifos prolonged the leaf selection time of L5, and reduced the number of binds per primary fold for L4 and L5. An LC 10 of chlorantraniliprole reduced the number of binds per primary fold for L4 and increased the number of head swings per bind for L5. An LC 10 of emamectin benzoate shortened the primary fold length for L5 and decreased the number of head swings per primary fold for L3 and L4 and the number of head swings per bind for L3, while an LC 25 of emamectin benzoate shortened the fold length per 24 h for L5 and folding time for L3. An LC 10 of spinosad lowered the fold length per 24 h and the number of head swings for L5. An LC 25 of spinosad prolonged leaf selection time, and decreased primary fold length, binds per primary fold, binds per fold and fold length per 24 h in L5. Emamectin benzoate and spinosad exerted stronger sublethal effects on the folding and spinning behavior of C. medinalis than chlorpyrifos and chlorantraniliprole. These results provide better understanding of the sublethal effects of interactions of insecticides on C. medinalis. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

PubMed Central

Kitzmüller, Claudia; Zulehner, Nora; Roulias, Anargyros; Briza, Peter; Ferreira, Fatima; Faé, Ingrid; Fischer, Gottfried F.; Bohle, Barbara

2015-01-01

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing. PMID:25670010

Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

PubMed Central

Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

2014-01-01

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

Testosterone during Pregnancy and Gender Role Behavior of Preschool Children: A Longitudinal, Population Study.

ERIC Educational Resources Information Center

Hines, Melissa; Golombok, Susan; Rust, John; Johnston, Katie J.; Golding, Jean

2002-01-01

Related blood levels of testosterone and sex hormone-binding globulin in pregnant women to gender role behavior among 342 male and 337 female offspring at 3.5 years. Found that testosterone levels related linearly to girls' gender role behavior. Neither hormone related to boys' gender role behavior. Other factors, including older brothers or…

Activation of serotonin 5-HT(2C) receptor suppresses behavioral sensitization and naloxone-precipitated withdrawal symptoms in heroin-treated mice.

PubMed

Wu, Xian; Pang, Gang; Zhang, Yong-Mei; Li, Guangwu; Xu, Shengchun; Dong, Liuyi; Stackman, Robert W; Zhang, Gongliang

2015-10-21

Abuse and dependence to heroin has evolved into a global epidemic as a significant clinical and societal problem with devastating consequences. Repeated exposure to heroin can induce long-lasting behavioral sensitization and withdrawal. Pharmacological activation of 5-HT2C receptors (5-HT2CRs) suppresses psychostimulant-induced drug-seeking and behavioral sensitization. The present study examined the effect of a selective 5-HT2CR agonist lorcaserin on behavioral sensitization and naloxone-precipitated withdrawal symptoms in heroin-treated mice. Male mice received heroin (1.0 mg/kg, s.c.) twice a day for 3 days and then drug treatment was suspended for 5 days. On day 9, a challenge dose of heroin (1.0 mg/kg) was administered to examine the expression of behavioral sensitization. Lorcaserin administered during the development, withdrawal or expression stage suppressed heroin-induced behavioral sensitization on day 9. Another cohort of mice received increasing doses of heroin over a 4.5-day period. Lorcaserin, or the positive control clonidine (an α2-adrenoceptor agonist) suppressed naloxone-precipitated withdrawal symptoms in heroin-treated mice. These findings suggest that activation of 5-HT2CRs suppresses behavioral sensitization and withdrawal in heroin-treated mice. Thus, pharmacological activation of 5-HT2CRs may represent a new avenue for the treatment of heroin addiction. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Activation of serotonin 5-HT2C receptor suppresses behavioral sensitization and naloxone-precipitated withdrawal symptoms in heroin-treated mice

PubMed Central

Li, Guangwu; Xu, Shengchun; Dong, Liuyi; Stackman, Robert W.; Zhang, Gongliang

2015-01-01

Abuse and dependence to heroin has evolved into a global epidemic as a significant clinical and societal problem with devastating consequences. Repeated exposure to heroin can induce long-lasting behavioral sensitization and withdrawal. Pharmacological activation of 5-HT2C receptors (5-HT2CRs) suppresses psychostimulant-induced drug-seeking and behavioral sensitization. The present study examined the effect of a selective 5-HT2CR agonist lorcaserin on behavioral sensitization and naloxone-precipitated withdrawal symptoms in heroin-treated mice. Male mice received heroin (1.0 mg/kg, s.c.) twice a day for 3 days and then drug treatment was suspended for 5 days. On day 9, a challenge dose of heroin (1.0 mg/kg) was administered to examine the expression of behavioral sensitization. Lorcaserin administered during the development, withdrawal or expression stage suppressed heroin-induced behavioral sensitization on day 9. Another cohort of mice received increasing doses of heroin over a 4.5-day period. Lorcaserin, or the positive control clonidine (an α2-adrenoceptor agonist) suppressed naloxone-precipitated withdrawal symptoms in heroin-treated mice. These findings suggest that activation of 5-HT2CRs suppresses behavioral sensitization and withdrawal in heroin-treated mice. Thus, pharmacological activation of 5-HT2CRs may represent a new avenue for the treatment of heroin addiction. PMID:26375926

Activation of serotonin 5-HT2C receptor suppresses behavioral sensitization and naloxone-precipitated withdrawal symptoms in morphine-dependent mice

PubMed Central

Zhang, Gongliang; Wu, Xian; Zhang, Yong-Mei; Liu, Huan; Jiang, Qin; Pang, Gang; Tao, Xinrong; Dong, Liuyi; Stackman, Robert W.

2015-01-01

Opioid abuse and dependence have evolved into an international epidemic as a significant clinical and social problem with devastating consequences. Repeated exposure to the opioid, for example morphine, can induce profound, long-lasting behavioral sensitization and physical dependence, which are thought to reflect neuroplasticity in neural circuitry. Central serotonin (5-HT) neurotransmission participates in the development of dependence on and the expression of withdrawal from morphine. Serotonin 5-HT2C receptor (5-HT2CR) agonists suppress psychostimulant nicotine or cocaine-induced behavioral sensitization and drug-seeking behavior; however, the impact of 5-HT2CR agonists on behaviors relevant to opioid abuse and dependence has not been reported. In the present study, the effects of 5-HT2CR activation on the behavioral sensitization and naloxone-precipitated withdrawal symptoms were examined in mice underwent repeated exposure to morphine. Male mice received morphine (10 mg/kg, s.c.) to develop behavioral sensitization. Lorcaserin, a 5-HT2CR agonist, prevented the induction and expression, but not the development, of morphine-induced behavioral sensitization. Another cohort of mice received increasing doses of morphine over a 7-day period to induce morphine-dependence. Pretreatment of lorcaserin, or the positive control clonidine (an alpha 2-adrenoceptor agonist), ameliorated the naloxone-precipitated withdrawal symptoms. SB 242084, a selective 5-HT2CR antagonist, prevented the lorcaserin-mediated suppression of behavioral sensitization and withdrawal. Chronic morphine treatment was associated with an increase in the expression of 5-HT2CR protein in the ventral tegmental area, locus coeruleus and nucleus accumbens. These findings suggest that 5-HT2CR can modulate behavioral sensitization and withdrawal in morphine-dependent mice, and the activation of 5-HT2CR may represent a new avenue for the treatment of opioid addiction. PMID:26432939

The relationship between water binding and desiccation tolerance in tissues

NASA Technical Reports Server (NTRS)

Vertucci, C. W.; Leopold, A. C.

1987-01-01

In an effort to define the nature of desiccation tolerance, a comparison of the water sorption characteristics was made between tissues that were resistant and tissues that were sensitive to desiccation. Water sorption isotherms were constructed for germinated and ungerminated soybean axes and also for fronds of several species of Polypodium with varying tolerance to dehydration. The strength of water binding was determined by van't Hoff as well as D'Arcy/Watt analyses of the isotherms at 5, 15, and/or 25 degrees C. Tissues which were sensitive to desiccation had a poor capacity to bind water tightly. Tightly bound water can be removed from soybean and pea seeds by equilibration at 35 degrees C over very low relative humidities; this results in a reduction in the viability of the seed. We suggest that region 1 water (i.e. water bound with very negative enthalpy values) is an important component of desiccation tolerance.

Increased /sup 3/H-spiperone binding sites in mesolimbic area related to methamphetamine-induced behavioral hypersensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akiyama, K.; Sato, M.; Otsuki, S.

1982-02-01

The specific /sup 3/H-spiperone binding to membrane homogenates of the striatum, mesolimbic area, and frontal cortex was examined in two groups of rats pretreated once daily with saline or 4 mg/kg of methamphetamine (MAP) for 14 days. At 7 days following cessation of chronic pretreatment, all rats received an injection of 4 mg/kg of MAP and were decapitated 1 hr after the injection. In the chronic saline-pretreatment group, the single administration of MAP induced significant changes in the number (Bmax) of specific /sup 3/H-spiperone binding sites (a decrease in the striatum and an increase in the mesolimbic area and frontalmore » cortex), but no significant changes in the affinity (KD) in any brain area. The chronic MAP pretreatment markedly augmented the changes in Bmax in the striatum and mesolimbic area. The increase in specific /sup 3/H-spiperone binding sites in the mesolimbic area is discussed in relation to MAP-induced behavioral hypersensitivity.« less

RNA buffers the phase separation behavior of prion-like RNA binding proteins.

PubMed

Maharana, Shovamayee; Wang, Jie; Papadopoulos, Dimitrios K; Richter, Doris; Pozniakovsky, Andrey; Poser, Ina; Bickle, Marc; Rizk, Sandra; Guillén-Boixet, Jordina; Franzmann, Titus M; Jahnel, Marcus; Marrone, Lara; Chang, Young-Tae; Sterneckert, Jared; Tomancak, Pavel; Hyman, Anthony A; Alberti, Simon

2018-05-25

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The γ-aminobutyric acid type B (GABAB) receptor agonist baclofen inhibits morphine sensitization by decreasing the dopamine level in rat nucleus accumbens.

PubMed

Fu, Zhenyu; Yang, Hongfa; Xiao, Yuqiang; Zhao, Gang; Huang, Haiyan

2012-07-10

Repeated morphine exposure can induce behavioral sensitization. There are evidences have shown that central gamma-aminobutyric acid (GABA) system is involved in morphine dependence. However, the effect of a GABAB receptor agonist baclofen on morphine-induced behavioral sensitization in rats is unclear. We used morphine-induced behavioral sensitization model in rat to investigate the effects of baclofen on behavioral sensitization. Moreover, dopamine release in the shell of the nucleus accumbens was evaluated using microdialysis assay in vivo. The present study demonstrated that morphine challenge (3 mg/kg, s.c.) obviously enhanced the locomotor activity following 4-day consecutive morphine administration and 3-day withdrawal period, which indicated the expression of morphine sensitization. In addition, chronic treatment with baclofen (2.5, 5 mg/kg) significantly inhibited the development of morphine sensitization. It was also found that morphine challenge 3 days after repeated morphine administration produced a significant increase of extracellular dopamine release in nucleus accumbens. Furthermore, chronic treatment with baclofen decreased the dopamine release induced by morphine challenge. Our results indicated that gamma-aminobutyric acid system plays an important role in the morphine sensitization in rat and suggested that behavioral sensitization is a promising model to study the mechanism underlying drug abuse.

Ca2+ sensitizers: An emerging class of agents for counterbalancing weakness in skeletal muscle diseases?

PubMed

Ochala, Julien

2010-02-01

Ca(2+) ions are key regulators of skeletal muscle contraction. By binding to contractile proteins, they initiate a cascade of molecular events leading to cross-bridge formation and ultimately, muscle shortening and force production. The ability of contractile proteins to respond to Ca(2+) attachment, also known as Ca(2+) sensitivity, is often compromised in acquired and congenital skeletal muscle disorders. It constitutes, undoubtedly, a major physiological cause of weakness for patients. In this review, we discuss recent studies giving strong molecular and cellular evidence that pharmacological modulators of some of the contractile proteins, also termed Ca(2+) sensitizers, are efficient agents to improve Ca(2+) sensitivity and function in diseased skeletal muscle cells. In fact, they compensate for the impaired contractile proteins response to Ca(2+) binding. Currently, such Ca(2+) sensitizing compounds are successfully used for reducing problems in cardiac disorders. Therefore, in the future, under certain conditions, these agents may represent an emerging class of agents to enhance the quality of life of patients suffering from skeletal muscle weakness. Copyright 2009 Elsevier B.V. All rights reserved.

Failure of enzyme encapsulation to prevent sensitization of workers in the dry bleach industry.

PubMed

Liss, G M; Kominsky, J R; Gallagher, J S; Melius, J; Brooks, S M; Bernstein, I L

1984-03-01

BDE added to dry bleach have been associated with immunologic sensitization and development of clinical allergic disease in detergent workers and occasionally in consumers. However, improved dust control and modification of the manufacturing process through encapsulation of enzyme were believed to have reduced or eliminated these problems. To determine whether or not immunologic sensitization could still develop in the detergent industry, we studied employees of a dry bleach manufacturing plant that incorporated encapsulated BDE into a consumer product. We performed air sampling for enzyme dust and total particulates, administered questionnaires, conducted physical examinations, and spirometry in 13 currently exposed, two previously exposed and nine nonexposed, employees. To assess sensitization status, RAST and ELISA were performed. Air concentrations of enzyme dust ranged from 0.002 to 1.57 micrograms/m3; all of these levels were below the TLV of 3.9 micrograms/m3. Positive BDE-specific RAST results (3.4%, 4.4%, and 8.0% binding) were obtained in three of 12 currently exposed workers. Results of personal breathing-zone air sampling indicated that these workers had high dust-exposure levels. Specificity of RAST was verified by RAST inhibition with BDE. BDE-RAST binding was not significantly elevated in the nonworkers (range: 0.6% to 1.4% binding). Positive results for specific IgG by ELISA were obtained in four of 12 currently exposed and in one of two previously exposed workers but in none of the nonexposed workers. We conclude that immunologic sensitization can develop after occupational exposure to encapsulated BDE in the dry bleach industry. We have not proved, however, that this immunologic reactivity is related to clinical sensitivity.

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

PubMed Central

Foster, David A.; Hantzopoulos, Petros; Zubay, Geoffrey

1982-01-01

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion. Images PMID:6809958

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

PubMed Central

Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

2009-01-01

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

Monoamine-Sensitive Developmental Periods Impacting Adult Emotional and Cognitive Behaviors

PubMed Central

Suri, Deepika; Teixeira, Cátia M; Cagliostro, Martha K Caffrey; Mahadevia, Darshini; Ansorge, Mark S

2015-01-01

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system development, such sensitive periods shape the formation of neurocircuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint as well as the environmental context. While allowing for adaptation, such sensitive periods are also vulnerability windows during which external and internal factors can confer risk to disorders by derailing otherwise resilient developmental programs. Here we review developmental periods that are sensitive to monoamine signaling and impact adult behaviors of relevance to psychiatry. Specifically, we review (1) a serotonin-sensitive period that impacts sensory system development, (2) a serotonin-sensitive period that impacts cognition, anxiety- and depression-related behaviors, and (3) a dopamine- and serotonin-sensitive period affecting aggression, impulsivity and behavioral response to psychostimulants. We discuss preclinical data to provide mechanistic insight, as well as epidemiological and clinical data to point out translational relevance. The field of translational developmental neuroscience has progressed exponentially providing solid conceptual advances and unprecedented mechanistic insight. With such knowledge at hand and important methodological innovation ongoing, the field is poised for breakthroughs elucidating the developmental origins of neuropsychiatric disorders, and thus understanding pathophysiology. Such knowledge of sensitive periods that determine the developmental trajectory of complex behaviors is a necessary step towards improving prevention and treatment approaches for neuropsychiatric disorders. PMID:25178408

Simultaneous ultramicroanalysis of both 17-keto-and 17beta-hydroxy androgens in biological fluids.

PubMed

Ganjam, V K

1976-11-01

Sensitive methods for quantifying androgens were lacking. Therefore, a relatively simple procedure for separating steroids was combined with highly specific assay methods so that eight androgens could be measured with high accuracy, precision and sensitivity. Semi-automated separations on Sephadex LH-20 columns used heptane:methylene chloride:ethanol:water (50:50:1:0.12) and a flow rate of 17.0 min/ml. The six peaks eluted contained androstenedine; androsterone, epiandrosterone and dihydrotestosterone; testosterone and dehydroepiandrosterone; 3alpha-androstanediol; 3beta-androstanediol; and androstenediol. Androstenedione, dehydroepiandrosterone and androstenediol were quantified using specific antisera (sensitivity less than or equal to 75 pg). Testosterone and dihydrotestosterone were measured by competitive protein-binding assays using rabbit TeBG (sensitivity less than or equal to 150 pg). 3alpha- and 3beta-androstanediol were similarly assayed using human TeBG (sensitivity approximately 150 pg). Androsterone was reduced with NaBH4 and the resulting 3alpha-androstanediol was assayed using human TeBG (sensitivity approximately 200 pg). Inter- and intra-assay variations were less than 10% for radioimmunoassays and less than 16% for competitive protein-binding assays over the entire dose response curve.

Visualisation of Collagen in fixed skeletal muscle tissue using fluorescently tagged Collagen binding protein CNA35.

PubMed

Mohammadkhah, Melika; Simms, Ciaran K; Murphy, Paula

2017-02-01

Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15μm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100μg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantification of [(11)C]yohimbine binding to α2 adrenoceptors in rat brain in vivo.

PubMed

Phan, Jenny-Ann; Landau, Anne M; Wong, Dean F; Jakobsen, Steen; Nahimi, Adjmal; Doudet, Doris J; Gjedde, Albert

2015-03-01

We quantified the binding potentials (BPND) of [(11)C]yohimbine binding in rat brain to alpha-2 adrenoceptors to evaluate [(11)C]yohimbine as an in vivo marker of noradrenergic neurotransmission and to examine its sensitivity to the level of noradrenaline. Dual [(11)C]yohimbine dynamic positron emission tomography (PET) recordings were applied to five Sprague Dawley rats at baseline, followed by acute amphetamine administration (2 mg/kg) to induce elevation of the endogenous level of noradrenaline. The volume of distribution (VT) of [(11)C]yohimbine was obtained using Logan plot with arterial plasma input. Because alpha-2 adrenoceptors are distributed throughout the brain, the estimation of the BPND is complicated by the absence of an anatomic region of no displaceable binding. We used the Inhibition plot to acquire the reference volume, VND, from which we calculated the BPND. Acute pharmacological challenge with amphetamine induced a significant decline of [(11)C]yohimbine BPND of ~38% in all volumes of interest. The BPND was greatest in the thalamus and striatum, followed in descending order by, frontal cortex, pons, and cerebellum. The experimental data demonstrate that [(11)C]yohimbine binding is sensitive to a challenge known to increase the extracellular level of noradrenaline, which can benefit future PET investigations of pathologic conditions related to disrupted noradrenergic neurotransmission.

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

PubMed

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Blunted Ambiguity Aversion During Cost-Benefit Decisions in Antisocial Individuals.

PubMed

Buckholtz, Joshua W; Karmarkar, Uma; Ye, Shengxuan; Brennan, Grace M; Baskin-Sommers, Arielle

2017-05-17

Antisocial behavior is often assumed to reflect aberrant risk processing. However, many of the most significant forms of antisocial behavior, including crime, reflect the outcomes of decisions made under conditions of ambiguity rather than risk. While risk and ambiguity are formally distinct and experimentally dissociable, little is known about ambiguity sensitivity in individuals who engage in chronic antisocial behavior. We used a financial decision-making task in a high-risk community-based sample to test for associations between sensitivity to ambiguity, antisocial behavior, and arrest history. Sensitivity to ambiguity was lower in individuals who met diagnostic criteria for Antisocial Personality Disorder. Lower ambiguity sensitivity was also associated with higher externalizing (but not psychopathy) scores, and with higher levels of aggression (but not rule-breaking). Finally, blunted sensitivity to ambiguity also predicted a greater frequency of arrests. Together, these data suggest that alterations in cost-benefit decision-making under conditions of ambiguity may promote antisocial behavior.

Preventive behaviors by the level of perceived infection sensitivity during the Korea outbreak of Middle East Respiratory Syndrome in 2015.

PubMed

Lee, Soon Young; Yang, Hee Jeong; Kim, Gawon; Cheong, Hae-Kwan; Choi, Bo Youl

2016-01-01

This study was performed to investigate the relationship between community residents' infection sensitivity and their levels of preventive behaviors during the 2015 Middle East Respiratory Syndrome (MERS) outbreak in Korea. Seven thousands two hundreds eighty one participants from nine areas in Gyeonggi-do including Pyeongtaek, the origin of the outbreak in 2015 agreed to participate in the survey and the data from 6,739 participants were included in the final analysis. The data on the perceived infection sensitivity were subjected to cluster analysis. The levels of stress, reliability/practice of preventive behaviors, hand washing practice and policy credibility during the outbreak period were analyzed for each cluster. Cluster analysis of infection sensitivity due to the MERS outbreak resulted in classification of participants into four groups: the non-sensitive group (14.5%), social concern group (17.4%), neutral group (29.1%), and overall sensitive group (39.0%). A logistic regression analysis found that the overall sensitive group with high sensitivity had higher stress levels (17.80; 95% confidence interval [CI], 13.77 to 23.00), higher reliability on preventive behaviors (5.81; 95% CI, 4.84 to 6.98), higher practice of preventive behaviors (4.53; 95% CI, 3.83 to 5.37) and higher practice of hand washing (2.71; 95% CI, 2.13 to 3.43) during the outbreak period, compared to the non-sensitive group. Infection sensitivity of community residents during the MERS outbreak correlated with gender, age, occupation, and health behaviors. When there is an outbreak in the community, there is need to maintain a certain level of sensitivity while reducing excessive stress, as well as promote the practice of preventive behaviors among local residents. In particular, target groups need to be notified and policies need to be established with a consideration of the socio-demographic characteristics of the community.

Evidence for specific annexin I-binding proteins on human monocytes.

PubMed Central

Goulding, N J; Pan, L; Wardwell, K; Guyre, V C; Guyre, P M

1996-01-01

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions. PMID:8687405

To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

PubMed

Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

2013-01-01

Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

Mouse strain differences in immobility and sensitivity to fluvoxamine and desipramine in the forced swimming test: analysis of serotonin and noradrenaline transporter binding.

PubMed

Sugimoto, Yumi; Kajiwara, Yoshinobu; Hirano, Kazufumi; Yamada, Shizuo; Tagawa, Noriko; Kobayashi, Yoshiharu; Hotta, Yoshihiro; Yamada, Jun

2008-09-11

Strain differences in immobility time in the forced swimming test were investigated in five strains of mice, namely, ICR, ddY, C57BL/6, DBA/2 and BALB/c mice. There were significant strain differences. The immobility times of ICR, ddY and C57BL/6 mice were longer than those of DBA/2 and BALB/c mice. Immobility times were not significantly related to locomotor activity in these strains. There were also differences in sensitivity to the selective serotonin reuptake inhibitor (SSRI) fluvoxamine. In ICR, ddY and C57BL/6 mice, fluvoxamine did not affect immobility time, while it reduced the immobility time of DBA/2 and BALB/c mice dose-dependently. The noradrenaline reuptake inhibitor desipramine decreased immobility time in all strains of mice. Serotonin (5-HT) transporter binding in the brains of all five strains of mice was also investigated. Analysis of 5-HT transporter binding revealed significant strain differences, being lower in DBA/2 and BALB/c mice than in other strains of mice. The amount of 5-HT transporter binding was correlated to baseline immobility time. However, there was no significant relation between noradrenaline transporter binding and immobility time. These results suggest that the duration of baseline immobility depends on the levels of 5-HT transporter binding, leading to apparent strain differences in immobility time in the forced swimming test. Furthermore, differences in 5-HT transporter binding may cause variations in responses to fluvoxamine.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

Aptamer-mediated colorimetric method for rapid and sensitive detection of chloramphenicol in food.

PubMed

Yan, Chao; Zhang, Jing; Yao, Li; Xue, Feng; Lu, Jianfeng; Li, Baoguang; Chen, Wei

2018-09-15

We report an aptamer-mediated colorimetric method for sensitive detection of chloramphenicol (CAP). The aptamer of CAP is immobilized by the hybridization with pre-immobilized capture probe in the microtiter plate. The horseradish peroxidase (HRP) is covalently attached to the aptamer by the biotin-streptavidin system for signal production. CAP will preferably bind with aptamer due to the high binding affinity, which attributes to the release of aptamer and HRP and thus, affects the optical signal intensity. Quantitative determination of CAP is successfully achieved in the wide range from 0.001 to 1000 ng/mL with detection limit of 0.0031 ng/mL, which is more sensitive than traditional immunoassays. This method is further validated by measuring the recovery of CAP spiked in two different food matrices (honey and fish). The aptamer-mediated colorimetric method can be a useful protocol for rapid and sensitive screening of CAP, and may be used as an alternative means for traditional immunoassays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

PubMed

Song, Myeong-Sub; Sekhon, Simranjeet Singh; Shin, Woo-Ri; Rhee, Sung-Keun; Ko, Jung Ho; Kim, Sang Yong; Min, Jiho; Ahn, Ji-Young; Kim, Yang-Hoon

2018-05-01

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Mutation of a Zinc-Binding Residue in the Glycine Receptor α1 Subunit Changes Ethanol Sensitivity In Vitro and Alcohol Consumption In Vivo

PubMed Central

McCracken, Lindsay M.; Blednov, Yuri A.; Trudell, James R.; Benavidez, Jillian M.; Betz, Heinrich

2013-01-01

Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs), which are potentiated by millimolar concentrations of ethanol. In addition, zinc ions also modulate GlyR function, and recent evidence suggests that physiologic concentrations of zinc enhance ethanol potentiation of GlyRs. Here, we first built a homology model of a zinc-bound GlyR using the D80 position as a coordination site for a zinc ion. Next, we investigated in vitro the effects of zinc on ethanol action at recombinant wild-type (WT) and mutant α1 GlyRs containing the D80A substitution, which eliminates zinc potentiation. At D80A GlyRs, the effects of 50 and 200 mM ethanol were reduced as compared with WT receptors. Also, in contrast to what was seen with WT GlyRs, neither adding nor chelating zinc changed the magnitude of ethanol enhancement of mutant D80A receptors. Next, we evaluated the in vivo effects of the D80A substitution by using heterozygous Glra1(D80A) knock-in (KI) mice. The KI mice showed decreased ethanol consumption and preference, and they displayed increased startle responses compared with their WT littermates. Other behavioral tests, including ethanol-induced motor incoordination and strychnine-induced convulsions, revealed no differences between the KI and WT mice. Together, our findings indicate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc binding at the D80 position may be important for mediating some of the behavioral effects of ethanol action at GlyRs. PMID:23230213

Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse.

PubMed

Walters, C L; Blendy, J A

2001-12-01

Addiction is a complex process that relies on the ability of an organism to integrate positive and negative properties of drugs of abuse. Therefore, studying the reinforcing as well as aversive components of drugs of abuse in a single model system will enable us to understand the role of final common mediators, such as cAMP response element-binding protein (CREB), in the addiction process. To this end, we analyzed mice with a mutation in the alpha and Delta isoforms of the CREB gene. Previously we have shown that CREB(alphaDelta) mutant mice in a mixed genetic background show attenuated signs of physical dependence, as measured by the classic signs of withdrawal. We have generated a uniform genetically stable F1 hybrid (129SvEv/C57BL/6) mouse line harboring the CREB mutation. We have found the functional activity of CREB in these F1 hybrid mice to be dramatically reduced compared with their wild-type littermates. These mice maintain a reduced withdrawal phenotype after chronic morphine. We are now poised to examine a number of complex behavioral phenotypes related to addiction in a well defined CREB-deficient mouse model. We demonstrate that the aversive properties of morphine are still present in CREB mutant mice despite a reduction of physical withdrawal. On the other hand, these mice do not respond to the reinforcing properties of morphine in a conditioned place preference paradigm. In contrast, CREB mutant mice demonstrate an enhanced response to the reinforcing properties of cocaine compared with their wild-type controls in both conditioned place preference and sensitization behaviors. These data may provide the first paradigm for differential vulnerability to various drugs of abuse.

Caudate neuronal recording in freely behaving animals following acute and chronic dose response methylphenidate exposure

PubMed Central

Claussen, Catherine M; Dafny, Nachum

2016-01-01

The misuse and abuse of the psychostimulant, methylphenidate (MPD) the drug of choice in the treatment of attention deficit hyperactivity disorder (ADHD) has seen a sharp uprising in recent years among both youth and adults for its cognitive enhancing effects and for recreational purposes. This uprise in illicit use has lead to many questions concerning the long term consequences of MPD exposure. The objective of this study was to record animal behavior concomitantly with the caudate nucleus (CN) neuronal activity following acute and repetitive (chronic) dose response exposure to methylphenidate (MPD). A saline control and three MPD dose (0.6, 2.5, and 10.0 mg/kg) groups were used. Behaviorally, the same MPD dose in some animals following chronic MPD exposure elicited behavioral sensitization and other animals elicited behavioral tolerance. Based on this finding, the CN neuronal population recorded from animals expressing behavioral sensitization were also evaluated separately from CN neurons recorded from animals expressing behavioral tolerance to chronic MPD exposure, respectively. Significant differences in CN neuronal population responses between the behaviorally sensitized and the behaviorally tolerant animals was observed for the 2.5 and 10.0 mg/kg MPD exposed groups. For 2.5 mg/kg MPD, behaviorally sensitized animals responded by decreasing their firing rates while behaviorally tolerant animals showed mainly an increase in their firing rates. The CN neuronal responses recorded from the behaviorally sensitized animals following 10.0 mg/kg MPD responded by increasing their firing rates whereas the CN neuronal recordings from the behaviorally tolerant animals showed that approximately half decreased their firing rates in response to 10.0 mg/kg MPD exposure. The comparison of percentage change in neuronal firing rates showed that the behaviorally tolerant animals trended to exhibit increases in their neuronal firing rates at ED1 following initial MPD exposure and oppositely at ED10 MPD rechallenge. While the behaviorally sensitized animals in general increased in their percentage change of firing rats were observed following acute 10.0 mg/kg MPD and the behaviorally sensitized 10.0 mg/kg MPD animals and a robust increase in neuronal firing rates at ED1 and ED10 rechallenge. These results suggest the need to first individually analyze animal behavioral activity, and than to evaluate the neuronal responses to the drug based on the animals behavioral response to chronic MPD exposure. PMID:26101057

Biological Applications of Extraordinary Electroconductance and Photovoltaic Effects in Inverse Extraordinary Optoconductance

NASA Astrophysics Data System (ADS)

Tran, Lauren Christine

The Extraordinary Electroconductance (EEC) sensor has been previously demonstrated to have an electric field sensitivity of 3.05V/cm in a mesoscopic-scale structure fabricated at the center of a parallel plate capacitor. In this thesis, we demonstrate the first successful application of EEC sensors as electrochemical detectors of protein binding and biological molecule concentration. Using the avidin derivative, captavidin, in complex with the vitamin biotin, the change in four-point measured resistance with fluid protein concentration of bare EEC sensors was shown to increase by a factor of four in the presence of biomolecular binding as compared to baseline. Calculations for approximate field strengths introduced by a bound captavidin molecule are also presented. The development of Inverse-Extraordinary Optoconductance (I-EOC), an effect which occurs in nanoscale sensors, is also discussed. In the I-EOC effect, electron transport transitions from ballistic to diffusive with increasing light intensity. In these novel, room temperature optical detectors, the resistance is low at low light intensity and resistance increases by 9462% in a 250nm device mesa upon full illumination with a 5 mW HeNe laser. This is the inverse of bulk and mesoscopic device behavior, in which resistance decreases with increasing photon density.

An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

NASA Technical Reports Server (NTRS)

Farrington, Marianne A.; Hymer, W. C.

1987-01-01

A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

NASA Technical Reports Server (NTRS)

Basel, L. E.; Cleland, R. E.

1992-01-01

A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

Rapid Recovery of Vesicular Dopamine Levels in Methamphetamine Users in Early Abstinence

PubMed Central

Boileau, Isabelle; McCluskey, Tina; Tong, Junchao; Furukawa, Yoshiaki; Houle, Sylvain; Kish, Stephen J

2016-01-01

We previously reported very low levels of dopamine in post-mortem striatum of chronic methamphetamine users, raising the possibility that restoration of normal dopamine levels could help in this addiction and perhaps prevent early relapse. To establish relevance of this finding to the living brain, we tested whether striatal [11C]-(+)-dihydrotetrabenazine binding, a vesicular monoamine transporter probe sensitive to changes in (stored) vesicular dopamine, is elevated in methamphetamine users. Chronic methamphetamine users underwent [11C]-(+)-dihydrotetrabenazine positron emission tomography scans during early (mean 2.6 days) and later (~10 days) abstinence. Striatal [11C]-(+)-dihydrotetrabenazine binding was elevated (suggesting low stored dopamine) in methamphetamine users (n=28; 2.6 days after last use) relative to controls (n=22) (+28%, p<0.0001) and correlated with severity and recency of drug use and with cognitive impairment and withdrawal symptoms. Mean [11C]-(+)-dihydrotetrabenazine binding levels in the subgroup of methamphetamine users who could remain abstinent ~10 days following last use (n=17) were normal at the follow-up scan. Our imaging data support post-mortem findings and suggest that chronic methamphetamine users have low brain levels of stored dopamine during very early abstinence from MA, which could contribute to behavioral and cognitive deficits. Findings also suggest a rapid recovery of stored dopamine in some methamphetamine users who become abstinent and who therefore might not benefit from dopamine replacement medication (eg, levodopa). Further study is necessary to establish whether those users who could not maintain abstinence for the second scan might have a more severe and persistent dopamine deficiency and who could benefit from this medication. PMID:26321315

Rapid Recovery of Vesicular Dopamine Levels in Methamphetamine Users in Early Abstinence.

PubMed

Boileau, Isabelle; McCluskey, Tina; Tong, Junchao; Furukawa, Yoshiaki; Houle, Sylvain; Kish, Stephen J

2016-03-01

We previously reported very low levels of dopamine in post-mortem striatum of chronic methamphetamine users, raising the possibility that restoration of normal dopamine levels could help in this addiction and perhaps prevent early relapse. To establish relevance of this finding to the living brain, we tested whether striatal [(11)C]-(+)-dihydrotetrabenazine binding, a vesicular monoamine transporter probe sensitive to changes in (stored) vesicular dopamine, is elevated in methamphetamine users. Chronic methamphetamine users underwent [(11)C]-(+)-dihydrotetrabenazine positron emission tomography scans during early (mean 2.6 days) and later (~10 days) abstinence. Striatal [(11)C]-(+)-dihydrotetrabenazine binding was elevated (suggesting low stored dopamine) in methamphetamine users (n=28; 2.6 days after last use) relative to controls (n=22) (+28%, p<0.0001) and correlated with severity and recency of drug use and with cognitive impairment and withdrawal symptoms. Mean [(11)C]-(+)-dihydrotetrabenazine binding levels in the subgroup of methamphetamine users who could remain abstinent ~10 days following last use (n=17) were normal at the follow-up scan. Our imaging data support post-mortem findings and suggest that chronic methamphetamine users have low brain levels of stored dopamine during very early abstinence from MA, which could contribute to behavioral and cognitive deficits. Findings also suggest a rapid recovery of stored dopamine in some methamphetamine users who become abstinent and who therefore might not benefit from dopamine replacement medication (eg, levodopa). Further study is necessary to establish whether those users who could not maintain abstinence for the second scan might have a more severe and persistent dopamine deficiency and who could benefit from this medication.

An active-site phenylalanine directs substrate binding and C-H cleavage in the alpha-ketoglutarate-dependent dioxygenase TauD.

PubMed

McCusker, Kevin P; Klinman, Judith P

2010-04-14

Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

Photonic crystal biosensor microplates with integrated fluid networks for high throughput applications in drug discovery

NASA Astrophysics Data System (ADS)

Choi, Charles J.; Chan, Leo L.; Pineda, Maria F.; Cunningham, Brian T.

2007-09-01

Assays used in pharmaceutical research require a system that can not only detect biochemical interactions with high sensitivity, but that can also perform many measurements in parallel while consuming low volumes of reagents. While nearly all label-free biosensor transducers to date have been interfaced with a flow channel, the liquid handling system is typically aligned and bonded to the transducer for supplying analytes to only a few sensors in parallel. In this presentation, we describe a fabrication approach for photonic crystal biosensors that utilizes nanoreplica molding to produce a network of sensors that are automatically self-aligned with a microfluidic network in a single process step. The sensor/fluid network is inexpensively produced on large surface areas upon flexible plastic substrates, allowing the device to be incorporated into standard format 96-well microplates. A simple flow scheme using hydrostatic pressure applied through a single control point enables immobilization of capture ligands upon a large number of sensors with 220 nL of reagent, and subsequent exposure of the sensors to test samples. A high resolution imaging detection instrument is capable of monitoring the binding within parallel channels at rates compatible with determining kinetic binding constants between the immobilized ligands and the analytes. The first implementation of this system is capable of monitoring the kinetic interactions of 11 flow channels at once, and a total of 88 channels within an integrated biosensor microplate in rapid succession. The system was initially tested to characterize the interaction between sets of proteins with known binding behavior.

Mechanics of composite actin networks: in vitro and cellular perspectives

NASA Astrophysics Data System (ADS)

Upadhyaya, Arpita

2014-03-01

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

Viral-templated gold/polypyrrole nanopeapods for an ammonia gas sensor

NASA Astrophysics Data System (ADS)

Yan, Yiran; Zhang, Miluo; Moon, Chung Hee; Su, Heng-Chia; Myung, Nosang V.; Haberer, Elaine D.

2016-08-01

One-dimensional gold/polypyrrole (Au/PPy) nanopeapods were fabricated using a viral template: M13 bacteriophage. The genetically modified filamentous virus displayed gold-binding peptides along its length, allowing selective attachment of gold nanoparticles (Au NPs) under ambient conditions. A PPy shell was electropolymerized on the viral-templated Au NP chains forming nanopeapod structures. The PPy shell morphology and thickness were controlled through electrodeposition potential and time, resulting in an ultra-thin conductive polymer shell of 17.4 ± 3.3 nm. A post-electrodeposition acid treatment was used to modify the electrical properties of these hybrid materials. The electrical resistance of the nanopeapods was monitored at each assembly step. Chemiresistive ammonia (NH3) gas sensors were developed from networks of the hybrid Au/PPy nanostructures. Room temperature sensing performance was evaluated from 5 to 50 ppmv and a mixture of reversible and irreversible chemiresistive behavior was observed. A sensitivity of 0.30%/ppmv was found for NH3 concentrations of 10 ppmv or less, and a lowest detection limit (LDL) of 0.007 ppmv was calculated. Furthermore, acid-treated devices exhibited an enhanced sensitivity of 1.26%/ppmv within the same concentration range and a calculated LDL of 0.005 ppmv.

Neurons as sensors: individual and cascaded chemical sensing.

PubMed

Prasad, Shalini; Zhang, Xuan; Yang, Mo; Ozkan, Cengiz S; Ozkan, Mihrimah

2004-07-15

A single neuron sensor has been developed based on the interaction of gradient electric fields and the cell membrane. Single neurons are rapidly positioned over individual microelectrodes using positive dielectrophoretic traps. This enables the continuous extracellular electrophysiological measurements from individual neurons. The sensor developed using this technique provides the first experimental method for determining single cell sensitivity; the speed of response and the associated physiological changes to a broad spectrum of chemical agents. Binding of specific chemical agents to a specific combination of receptors induces changes to the extracellular membrane potential of a single neuron, which can be translated into unique "signature patterns" (SP), which function as identification tags. Signature patterns are derived using Fast Fourier Transformation (FFT) analysis and Wavelet Transformation (WT) analysis of the modified extracellular action potential. The validity and the sensitivity of the system are demonstrated for a variety of chemical agents ranging from behavior altering chemicals (ethanol), environmentally hazardous agents (hydrogen peroxide, EDTA) to physiologically harmful agents (pyrethroids) at pico- and femto-molar concentrations. The ability of a single neuron to selectively identify specific chemical agents when injected in a serial manner is demonstrated in "cascaded sensing".

Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1

PubMed Central

Hota, Prasanta K; Buck, Matthias

2009-01-01

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation. PMID:19388051

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using /sup 125/I-alpha scorpion toxin. I. Preferential labeling of glial cells on the presynaptic side

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudier, J.L.; Jover, E.; Cau, P.

1988-05-01

Alpha-scorpion toxins bind specifically to the voltage-sensitive sodium channel in excitable membranes, and binding is potential-dependent. The radioiodinated toxin II from the scorpion Androctonus australis Hector (alpha ScTx) was used to localize voltage-sensitive sodium channels on the presynaptic side of mouse neuromuscular junctions (NMJ) by autoradiography using both light and electron microscopy. Silver grain localization was analyzed by the cross-fire method. At the light-microscopic level, grain density over NMJ appeared 6-8x higher than over nonjunctional muscle membrane. The specificity of labeling was verified by competition/displacement with an excess of native alpha ScTx. Labeling was also inhibited by incubation in depolarizingmore » conditions, showing its potential-dependence. At the electron-microscopic level, analysis showed that voltage-sensitive sodium channels labeled with alpha ScTx were almost exclusively localized on membranes, as expected. Due to washout after incubation, appreciable numbers of binding sites were not found on the postsynaptic membranes. However, on the presynaptic side, alpha ScTx-labeled voltage-sensitive sodium channels were localized on the membrane of non-myelin-forming Schwann cells covering NMJ. The axonal presynaptic membrane was not labeled. These results show that voltage-sensitive sodium channels are present on glial cells in vivo, as already demonstrated in vitro. It is proposed that these glial channels could be indirectly involved in the ionic homeostasis of the axonal environment.« less

NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

PubMed

Baldwin, Amy; Pirisi, Lucia; Creek, Kim E

2004-04-01

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

Direct sensing of fluoride in aqueous solutions using a boronic acid based sensor.

PubMed

Wu, Xin; Chen, Xuan-Xuan; Song, Bing-Nan; Huang, Yan-Jun; Ouyang, Wen-Juan; Li, Zhao; James, Tony D; Jiang, Yun-Bao

2014-11-21

Binding of the fluoride ion triggers aggregation of a pyreneboronic acid-catechol ensemble in acidic aqueous solutions, giving rise to intense excimer emission, allowing for sensitive fluoride ion sensing at ppm levels, with an apparent fluoride binding constant higher than 10(3) M(-1) which is unprecedented for boronic acid sensors in water.

Native Mass Spectrometry in Fragment-Based Drug Discovery.

PubMed

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

PubMed Central

Okuno, Daichi; Nishiyama, Masayoshi; Noji, Hiroyuki

2013-01-01

F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å3 and +88 Å3 for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding. PMID:24094404

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lowery, Thomas J.; Doucleff, Michealeen; Ruiz, E. Janette

2005-02-01

The chemical shift of the {sup 129}Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins (Rubin et al. 2000; Lowery et al. 2004). Here we show that the {sup 129}Xe shift can sense more subtle changes: magnesium binding, BeF{sub 3}{sup -} activation, and peptide binding by the E. coli chemotaxis Y protein. {sup 1}H-{sup 15}N correlation spectroscopy and x-ray crystallography were used to identify two xenon-binding cavities in CheY that are primarily responsible for the shiftmore » changes. One site is near the active site, and the other is near the peptide binding site.« less

Identification of 50- and 23-/25-kDa HeLa cell membrane glycoproteins involved in poliovirus infection: occurrence of poliovirus specific binding sites on susceptible and nonsusceptible cells.

PubMed

Barnert, R H; Zeichhardt, H; Habermehl, K O

1992-02-01

Glycoproteins in the range 50 and 23/25 kDa were identified as poliovirus specific binding sites on HeLa cells with the monoclonal antibody mAb 122. mAb 122 is characterized by its partial inhibiting effect on poliovirus reproduction and adsorption when prebound to HeLa cells. The binding sites are endocytosed in native cells and specific for poliovirus as mAb 122 did not interfere with the adsorption of human rhinovirus type 14 (HRV 14). The poliovirus binding sites are present also on nonprimate so called nonsusceptible cells, e.g., mouse L-cells, as could be shown with sensitive ELISA based binding assays and performance of binding studies with fixed cells at 37 degrees.

In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

PubMed

Aasa-Chapman, Marlén M I; Cheney, Kelly M; Hué, Stéphane; Forsman, Anna; O'Farrell, Stephen; Pellegrino, Pierre; Williams, Ian; McKnight, Áine

2011-01-01

The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

Selective regulation of YB-1 mRNA translation by the mTOR signaling pathway is not mediated by 4E-binding protein.

PubMed

Lyabin, D N; Ovchinnikov, L P

2016-03-02

The Y-box binding protein 1 (YB-1) is a key regulator of gene expression at the level of both translation and transcription. The mode of its action on cellular events depends on its subcellular distribution and the amount in the cell. So far, the regulatory mechanisms of YB-1 synthesis have not been adequately studied. Our previous finding was that selective inhibition of YB-1 mRNA translation was caused by suppression of activity of the mTOR signaling pathway. It was suggested that this event may be mediated by phosphorylation of the 4E-binding protein (4E-BP). Here, we report that 4E-BP alone can only slightly inhibit YB-1 synthesis both in the cell and in vitro, although it essentially decreases binding of the 4F-group translation initiation factors to mRNA. With inhibited mTOR kinase, the level of mRNA binding to the eIF4F-group factors was decreased, while that to 4E-BP1 was increased, as was observed for both mTOR kinase-sensitive mRNAs and those showing low sensitivity. This suggests that selective inhibition of translation of YB-1 mRNA, and probably some other mRNAs as well, by mTOR kinase inhibitors is not mediated by the action of the 4E-binding protein upon functions of the 4F-group translation initiation factors.

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.

2013-01-01

Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

Characterization of strychnine-sensitive glycine receptor in the intact frog retina: modulation by protein kinases.

PubMed

Salceda, Rocío; Aguirre-Ramirez, Marisela

2005-03-01

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

Object-Location Memory: A Lesion-Behavior Mapping Study in Stroke Patients

ERIC Educational Resources Information Center

van Asselen, Marieke; Kessels, Roy P. C.; Frijns, Catharina J. M.; Kappelle, L. Jaap; Neggers, Sebastiaan F. W.; Postma, Albert

2009-01-01

Object-location memory is an important form of spatial memory, comprising different subcomponents that each process specific types of information within memory, i.e. remembering objects, remembering positions and binding these features in memory. In the current study we investigated the neural correlates of binding categorical (relative) or…

Cellular level models as tools for cytokine design.

PubMed

Radhakrishnan, Mala L; Tidor, Bruce

2010-01-01

Cytokines and growth factors are critical regulators that connect intracellular and extracellular environments through binding to specific cell-surface receptors. They regulate a wide variety of immunological, growth, and inflammatory response processes. The overall signal initiated by a population of cytokine molecules over long time periods is controlled by the subtle interplay of binding, signaling, and trafficking kinetics. Building on the work of others, we abstract a simple kinetic model that captures relevant features from cytokine systems as well as related growth factor systems. We explore a large range of potential biochemical behaviors, through systematic examination of the model's parameter space. Different rates for the same reaction topology lead to a dramatic range of biochemical network properties and outcomes. Evolution might productively explore varied and different portions of parameter space to create beneficial behaviors, and effective human therapeutic intervention might be achieved through altering network kinetic properties. Quantitative analysis of the results reveals the basis for tensions among a number of different network characteristics. For example, strong binding of cytokine to receptor can increase short-term receptor activation and signal initiation but decrease long-term signaling due to internalization and degradation. Further analysis reveals the role of specific biochemical processes in modulating such tensions. For instance, the kinetics of cytokine binding and receptor activation modulate whether ligand-receptor dissociation can generally occur before signal initiation or receptor internalization. Beyond analysis, the same models and model behaviors provide an important basis for the design of more potent cytokine therapeutics by providing insight into how binding kinetics affect ligand potency. (c) 2010 American Institute of Chemical Engineers

Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

PubMed

Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

2016-02-01

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals. Copyright © 2015 John Wiley & Sons, Ltd.

The gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen inhibits morphine sensitization by decreasing the dopamine level in rat nucleus accumbens

PubMed Central

2012-01-01

Background Repeated morphine exposure can induce behavioral sensitization. There are evidences have shown that central gamma-aminobutyric acid (GABA) system is involved in morphine dependence. However, the effect of a GABAB receptor agonist baclofen on morphine-induced behavioral sensitization in rats is unclear. Methods We used morphine-induced behavioral sensitization model in rat to investigate the effects of baclofen on behavioral sensitization. Moreover, dopamine release in the shell of the nucleus accumbens was evaluated using microdialysis assay in vivo. Results The present study demonstrated that morphine challenge (3 mg/kg, s.c.) obviously enhanced the locomotor activity following 4-day consecutive morphine administration and 3-day withdrawal period, which indicated the expression of morphine sensitization. In addition, chronic treatment with baclofen (2.5, 5 mg/kg) significantly inhibited the development of morphine sensitization. It was also found that morphine challenge 3 days after repeated morphine administration produced a significant increase of extracellular dopamine release in nucleus accumbens. Furthermore, chronic treatment with baclofen decreased the dopamine release induced by morphine challenge. Conclusions Our results indicated that gamma-aminobutyric acid system plays an important role in the morphine sensitization in rat and suggested that behavioral sensitization is a promising model to study the mechanism underlying drug abuse. PMID:22559224

The Interaction Between Punishment Sensitivity and Effortful Control for Emerging Adults' Substance Use Behaviors.

PubMed

Kahn, Rachel E; Chiu, Pearl H; Deater-Deckard, Kirby; Hochgraf, Anna K; King-Casas, Brooks; Kim-Spoon, Jungmeen

2018-01-08

Within the dual systems perspective, high reward sensitivity and low punishment sensitivity in conjunction with deficits in cognitive control may contribute to high levels of risk taking, such as substance use. The current study examined whether the individual components of effortful control (inhibitory control, attentional control, and activation control) serve as regulators and moderate the association between reward or punishment sensitivity and substance use behaviors. A total of 1,808 emerging adults from a university setting (Mean age = 19.48; 72% female) completed self-report measures of reward and punishment sensitivity, effortful control, and substance use. Findings indicated significant two-way interactions for punishment sensitivity and inhibitory control for alcohol and marijuana use. The form of these interactions revealed a significant negative association between punishment sensitivity and alcohol and marijuana use at low levels of inhibitory control. No significant interactions emerged for reward sensitivity or other components of effortful control. The current findings provide preliminary evidence suggesting the dual systems theorized to influence risk taking behavior interact to make joint contributions to health risk behaviors such as substance use in emerging adults.

Emotional Impairment and Persistent Upregulation of mGlu5 Receptor following Morphine Abstinence: Implications of an mGlu5-MOPr Interaction

PubMed Central

Zanos, Panos; Georgiou, Polymnia; Gonzalez, Loreto Rojo; Hourani, Susanna; Chen, Ying; Kitchen, Ian; Kieffer, Brigitte L; Winsky-Sommerer, Raphaelle

2016-01-01

Background: A difficult problem in treating opioid addicts is the maintenance of a drug-free state because of the negative emotional symptoms associated with withdrawal, which may trigger relapse. Several lines of evidence suggest a role for the metabotropic glutamate receptor 5 in opioid addiction; however, its involvement during opioid withdrawal is not clear. Methods: Mice were treated with a 7-day escalating-dose morphine administration paradigm. Following withdrawal, the development of affective behaviors was assessed using the 3-chambered box, open-field, elevated plus-maze and forced-swim tests. Metabotropic glutamate receptor 5 autoradiographic binding was performed in mouse brains undergoing chronic morphine treatment and 7 days withdrawal. Moreover, since there is evidence showing direct effects of opioid drugs on the metabotropic glutamate receptor 5 system, the presence of an metabotropic glutamate receptor 5/μ-opioid receptor interaction was assessed by performing metabotropic glutamate receptor 5 autoradiographic binding in brains of mice lacking the μ-opioid receptor gene. Results: Withdrawal from chronic morphine administration induced anxiety-like, depressive-like, and impaired sociability behaviors concomitant with a marked upregulation of metabotropic glutamate receptor 5 binding. Administration of the metabotropic glutamate receptor 5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine, reversed morphine abstinence-induced depressive-like behaviors. A brain region-specific increase in metabotropic glutamate receptor 5 binding was observed in the nucleus accumbens shell, thalamus, hypothalamus, and amygdala of μ-opioid receptor knockout mice compared with controls. Conclusions: These results suggest an association between metabotropic glutamate receptor 5 alterations and the emergence of opioid withdrawal-related affective behaviors. This study supports metabotropic glutamate receptor 5 system as a target for the development of pharmacotherapies for the treatment of opioid addiction. Moreover, our data show direct effects of μ-opioid receptor system manipulation on metabotropic glutamate receptor 5 binding in the brain. PMID:26861145

Behavioral and auditory evoked potential audiograms of a false killer whale (Pseudorca crassidens)

NASA Astrophysics Data System (ADS)

Yuen, Michelle M. L.; Nachtigall, Paul E.; Breese, Marlee; Supin, Alexander Ya.

2005-10-01

Behavioral and auditory evoked potential (AEP) audiograms of a false killer whale were measured using the same subject and experimental conditions. The objective was to compare and assess the correspondence of auditory thresholds collected by behavioral and electrophysiological techniques. Behavioral audiograms used 3-s pure-tone stimuli from 4 to 45 kHz, and were conducted with a go/no-go modified staircase procedure. AEP audiograms used 20-ms sinusoidally amplitude-modulated tone bursts from 4 to 45 kHz, and the electrophysiological responses were received through gold disc electrodes in rubber suction cups. The behavioral data were reliable and repeatable, with the region of best sensitivity between 16 and 24 kHz and peak sensitivity at 20 kHz. The AEP audiograms produced thresholds that were also consistent over time, with range of best sensitivity from 16 to 22.5 kHz and peak sensitivity at 22.5 kHz. Behavioral thresholds were always lower than AEP thresholds. However, AEP audiograms were completed in a shorter amount of time with minimum participation from the animal. These data indicated that behavioral and AEP techniques can be used successfully and interchangeably to measure cetacean hearing sensitivity.

Behavioral profiles of feline breeds in Japan.

PubMed

Takeuchi, Yukari; Mori, Yuji

2009-08-01

To clarify the behavioral profiles of 9 feline purebreds, 2 Persian subbreeds and the Japanese domestic cat, a questionnaire survey was distributed to 67 small-animal veterinarians. We found significant differences among breeds in all behavioral traits examined except for "inappropriate elimination". In addition, sexual differences were observed in certain behaviors, including "aggression toward cats", "general activity", "novelty-seeking", and "excitability". These behaviors were more common in males than females, whereas "nervousness" and "inappropriate elimination" were rated higher in females. When all breeds were categorized into four groups on the basis of a cluster analysis using the scores of two behavioral trait factors called "aggressiveness/sensitivity" and "vivaciousness", the group including Abyssinian, Russian Blue, Somali, Siamese, and Chinchilla breeds showed high aggressiveness/sensitivity and low vivaciousness. In contrast, the group including the American Shorthair and Japanese domestic cat displayed low aggressiveness/sensitivity and high vivaciousness, and the Himalayan and Persian group showed mild aggressiveness/sensitivity and very low vivaciousness. Finally, the group containing Maine Coon, Ragdoll, and Scottish Fold breeds displayed very low aggressiveness/sensitivity and low vivaciousness. The present results demonstrate that some feline behavioral traits vary by breed and/or sex.

Social reward and social punishment sensitivity in relation to dietary restraint and binge/purge symptoms.

PubMed

Fussner, Lauren M; Luebbe, Aaron M; Smith, April R

2018-08-01

Disordered eating symptoms are associated with disrupted sensitivity to reward and punishment, broadly assessed. However, it is unknown how eating pathology is related to sensitivity to social reward and social punishment specifically. Drawing on Reinforcement Sensitivity Theory, the current study utilized a multi-method design to test whether disordered eating symptoms, specifically dietary restraint (DR) and binge/purge (BP), were similarly or uniquely related to sensitivity to social punishment and social reward. Female university students (N = 110, M = 18.66, SD = 0.89) completed self-report measures and a novel behavioral task measuring willingness to work for or to avoid social feedback. DR and BP symptoms were related to increased self-reported and behavioral sensitivity to social punishment, yet only when symptoms were tested in isolation. DR was associated with increased sensitivity to social reward across self-report and behavioral paradigms. BP symptoms were uniquely and positively related to self-reported sensitivity to social reward, but decreased behavioral sensitivity to social reward. Findings suggest that sensitivity to punishment may be a common factor related to DR and BP, whereas sensitivity to social reward may be a key factor differentiating disordered eating symptoms. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Novel Energetic Perchlorate Amine Salt: Synthesis, Properties, and Density Functional Theory Calculation

NASA Astrophysics Data System (ADS)

Ma, Peng; Pan, Yong; Jiang, Juncheng; Zhu, Shunguan

2017-10-01

A novel explosive, ethylenediamine triethylenediamine tetraperchlorate (ETT), was synthesized by a rapid " one-pot" method. The molecular and crystal structures of ETT were determined by X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The purity of the ETT was characterized by hydrogen nuclear magnetic resonance (H-NMR) spectra and elemental analysis (EA). The chemical and physical properties of the co-crystal ETT were further explored including impact sensitivity, velocity of detonation, and thermal behavior. The impact sensitivity of the ETT (h50% = 9.50 cm) is much lower than that of its components, ethylenediamine diperchlorate (ED) (h50% = 5.60 cm) and triethylenediamine diperchlorate (TD) (h50% = 2.10 cm). The measured detonation velocity is 8956 m/s (ρ = 1.873 g/cm3), which is much higher than that of TNT (6900 m/s) or RDX (8350 m/s). The co-crystal ETT shows a unique thermal behavior with a decomposition peak temperature at 365 °C. Band structure and density of states (DOS) of the ETT were confirmed by the CASTEP code. The first-principles tight-binding method within the general gradient approximation (GGA) was employed to study the electronic band structure as well as the DOS and Fermi energy. Hirshfeld surfaces were applied to analyze the intermolecular interactions in the co-crystal, and the results showed that weak interaction was dominantly mediated by H … O hydrogen bond. By analyzing the bond length at different temperatures, N-H covalent bond is the trigger bond for the ETT.

A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

PubMed

Fay, Jonathan F; Farrens, David L

2012-09-28

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation, and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed on the pathway to full receptor activation.

Memory Meets Control in Hippocampal and Striatal Binding of Stimuli, Responses, and Attentional Control States

PubMed Central

Brashier, Nadia M.

2015-01-01

The human brain encodes experience in an integrative fashion by binding together the various features of an event (i.e., stimuli and responses) into memory “event files.” A subsequent reoccurrence of an event feature can then cue the retrieval of the memory file to “prime” cognition and action. Intriguingly, recent behavioral studies indicate that, in addition to linking concrete stimulus and response features, event coding may also incorporate more abstract, “internal” event features such as attentional control states. In the present study, we used fMRI in healthy human volunteers to determine the neural mechanisms supporting this type of holistic event binding. Specifically, we combined fMRI with a task protocol that dissociated the expression of event feature-binding effects pertaining to concrete stimulus and response features, stimulus categories, and attentional control demands. Using multivariate neural pattern classification, we show that the hippocampus and putamen integrate event attributes across all of these levels in conjunction with other regions representing concrete-feature-selective (primarily visual cortex), category-selective (posterior frontal cortex), and control demand-selective (insula, caudate, anterior cingulate, and parietal cortex) event information. Together, these results suggest that the hippocampus and putamen are involved in binding together holistic event memories that link physical stimulus and response characteristics with internal representations of stimulus categories and attentional control states. These bindings then presumably afford shortcuts to adaptive information processing and response selection in the face of recurring events. SIGNIFICANCE STATEMENT Memory binds together the different features of our experience, such as an observed stimulus and concurrent motor responses, into so-called event files. Recent behavioral studies suggest that the observer's internal attentional state might also become integrated into the event memory. Here, we used fMRI to determine the brain areas responsible for binding together event information pertaining to concrete stimulus and response features, stimulus categories, and internal attentional control states. We found that neural signals in the hippocampus and putamen contained information about all of these event attributes and could predict behavioral priming effects stemming from these features. Therefore, medial temporal lobe and dorsal striatum structures appear to be involved in binding internal control states to event memories. PMID:26538657

Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies

PubMed Central

Pandya, Anshul. A.; Yakel, Jerrel L.

2013-01-01

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4β2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs. PMID:23732296

Structure of a peptide adsorbed on graphene and graphite.

PubMed

Katoch, Jyoti; Kim, Sang Nyon; Kuang, Zhifeng; Farmer, Barry L; Naik, Rajesh R; Tatulian, Suren A; Ishigami, Masa

2012-05-09

Noncovalent functionalization of graphene using peptides is a promising method for producing novel sensors with high sensitivity and selectivity. Here we perform atomic force microscopy, Raman spectroscopy, infrared spectroscopy, and molecular dynamics simulations to investigate peptide-binding behavior to graphene and graphite. We studied a dodecamer peptide identified with phage display to possess affinity for graphite. Optical spectroscopy reveals that the peptide forms secondary structures both in powder form and in an aqueous medium. The dominant structure in the powder form is α-helix, which undergoes a transition to a distorted helical structure in aqueous solution. The peptide forms a complex reticular structure upon adsorption on graphene and graphite, having a helical conformation different from α-helix due to its interaction with the surface. Our observation is consistent with our molecular dynamics calculations, and our study paves the way for rational functionalization of graphene using biomolecules with defined structures and, therefore, functionalities.

Detection and size analysis of proteins with switchable DNA layers.

PubMed

Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2009-04-01

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

Scaling Laws for NanoFET Sensors

NASA Astrophysics Data System (ADS)

Wei, Qi-Huo; Zhou, Fu-Shan

2008-03-01

In this paper, we report our numerical studies of the scaling laws for nanoplate field-effect transistor (FET) sensors by simplifying the nanoplates as random resistor networks. Nanowire/tube FETs are included as the limiting cases where the device width goes small. Computer simulations show that the field effect strength exerted by the binding molecules has significant impact on the scaling behaviors. When the field effect strength is small, nanoFETs have little size and shape dependence. In contrast, when the field-effect strength becomes stronger, there exists a lower detection threshold for charge accumulation FETs and an upper detection threshold for charge depletion FET sensors. At these thresholds, the nanoFET devices undergo a transition between low and large sensitivities. These thresholds may set the detection limits of nanoFET sensors. We propose to eliminate these detection thresholds by employing devices with very short source-drain distance and large width.

Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

PubMed

Ferguson, Angelica; Wang, Leyi; Altman, Roger B; Terry, Daniel S; Juette, Manuel F; Burnett, Benjamin J; Alejo, Jose L; Dass, Randall A; Parks, Matthew M; Vincent, C Theresa; Blanchard, Scott C

2015-11-05

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional dynamics within the human ribosome regulate the rate of active protein synthesis

PubMed Central

Ferguson, Angelica; Wang, Leyi; Altman, Roger B.; Terry, Daniel S.; Juette, Manuel F.; Burnett, Benjamin J.; Alejo, Jose L.; Dass, Randall A.; Parks, Matthew M.; Vincent, Theresa C.; Blanchard, Scott C.

2015-01-01

SUMMARY The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule FRET methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. PMID:26593721

Comparison of functional assays used in the clinical development of a placental malaria vaccine.

PubMed

Pehrson, Caroline; Heno, Kristine K; Adams, Yvonne; Resende, Mafalda; Mathiesen, Line; Soegaard, Max; de Jongh, Willem A; Theander, Thor G; Salanti, Ali; Nielsen, Morten A

2017-01-23

Malaria in pregnancy is associated with significant morbidity in pregnant women and their offspring. Plasmodium falciparum infected erythrocytes (IE) express VAR2CSA that mediates binding to chondroitin sulphate A (CSA) in the placenta. Two VAR2CSA-based vaccines for placental malaria are in clinical development. The purpose of this study was to evaluate the robustness and comparability of binding inhibition assays used in the clinical development of placental malaria vaccines. The ability of sera from animals immunised with different VAR2CSA constructs to inhibit IE binding to CSA was investigated in three in vitro assays using 96-well plates, petri dishes, capillary flow and an ex vivo placental perfusion assay. The inter-assay variation was not uniform between assays and ranged from above ten-fold in the flow assay to two-fold in the perfusion assay. The intra-assay variation was highest in the petri dish assay. A positive correlation between IE binding avidity and the level of binding after antibody inhibition in the petri dish assay indicate that high avidity IE binding is more difficult to inhibit. The highest binding inhibition sensitivity was found in the 96-well and petri dish assays compared to the flow and perfusion assays where binding inhibition required higher antibody titers. The inhibitory capacity of antibodies is not easily translated between assays and the high sensitivity of the 96-well and petri dish assays stresses the need for comparing serial dilutions of serum. Furthermore, IE binding avidity must be in the same range when comparing data from different days. There was an overall concordance in the capacity of antibody-mediated inhibition, when comparing the in vitro assays with the perfusion assay, which more closely represents in vivo conditions. Importantly the ID1-ID2a protein in a liposomal formulation, currently in a phase I trial, effectively induced antibodies that inhibited IE adhesion in placental tissue. Copyright © 2016. Published by Elsevier Ltd.

Immobilization of heparin/poly-(L)-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior.

PubMed

Liu, Tao; Liu, Yang; Chen, Yuan; Liu, Shihui; Maitz, Manfred F; Wang, Xue; Zhang, Kun; Wang, Jian; Wang, Yuan; Chen, Junying; Huang, Nan

2014-05-01

Restenosis, thrombosis formation and delayed endothelium regeneration continue to be problematic for coronary artery stent therapy. To improve the hemocompatibility of the cardiovascular implants and selectively direct vascular cell behavior, a novel kind of heparin/poly-l-lysine (Hep/PLL) nanoparticle was developed and immobilized on a dopamine-coated surface. The stability and structural characteristics of the nanoparticles changed with the Hep:PLL concentration ratio. A Hep density gradient was created on a surface by immobilizing nanoparticles with various Hep:PLL ratios on a dopamine-coated surface. Antithrombin III binding quantity was significantly enhanced, and in plasma the APTT and TT times as coagulation tests were prolonged, depending on the Hep density. A low Hep density is sufficient to prevent platelet adhesion and activation. The sensitivity of vascular cells to the Hep density is very different: high Hep density inhibits the growth of all vascular cells, while low Hep density could selectively inhibit smooth muscle cell hyperplasia but promote endothelial progenitor cells and endothelial cell proliferation. These observations provide important guidance for modification of surface heparinization. We suggest that this method will provide a potential means to construct a suitable platform on a stent surface for selective direction of vascular cell behavior with low side effects. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Socialization of prosocial behavior: Gender differences in the mediating role of child brain volume.

PubMed

Kok, Rianne; Prinzie, Peter; Bakermans-Kranenburg, Marian J; Verhulst, Frank C; White, Tonya; Tiemeier, Henning; van IJzendoorn, Marinus H

2018-08-01

Evidence has been accumulating for the impact of normal variation in caregiving quality on brain morphology in children, but the question remains whether differences in brain volume related to early caregiving translate to behavioral implications. In this longitudinal population-based study (N = 162), moderated mediation was tested for the relation between parental sensitivity and child prosocial behavior via brain volume, in boys and girls. Both maternal and paternal sensitivity were repeatedly observed between 1 and 4 years of age. Brain volume was assessed using magnetic resonance imaging measurements at age 8, and self-reported prosocial behavior of children was assessed at 9 years of age. Parental sensitivity was positively related to child brain volume, and to child prosocial behavior at trend level. Child brain volume was negatively related to child prosocial behavior. A significant gender-by-brain interaction was found, illustrating that daughters of sensitive parents were more prosocial and that less prosocial behavior was reported for girls with a larger total brain volume. Child gender significantly moderated the indirect effect of parental sensitivity on prosocial behavior via total brain volume. A significant indirect pathway was found only in girls. The results warrant replication but indicate the importance of considering gender when studying the behavioral implications of differences in brain volume related to early caregiving experiences.

Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse.

PubMed

Mousseau, D D; Larson, A A

1994-09-01

We have previously observed similarities in the behavioral effects produced by the NH2-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl)-guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro2, D-Phe7]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [3H]-DTG binding sites in the mouse brain and spinal cord in vitro: Brain; DTG = HAL > SP = MOR = NAL > SP(1-7) > D-SP(1-7) > SP(5-11): Spinal cord; DTG = HAL > SP(1-7) = MOR = NAL > SP > D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [3H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1-7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site.

Quantum transport in alkane molecular wires: Effects of binding modes and anchoring groups

NASA Astrophysics Data System (ADS)

Sheng, W.; Li, Z. Y.; Ning, Z. Y.; Zhang, Z. H.; Yang, Z. Q.; Guo, H.

2009-12-01

Effects of binding modes and anchoring groups on nonequilibrium electronic transport properties of alkane molecular wires are investigated from atomic first-principles based on density functional theory and nonequilibrium Green's function formalism. Four typical binding modes, top, bridge, hcp-hollow, and fcc-hollow, are considered at one of the two contacts. For wires with three different anchoring groups, dithiol, diamine, or dicarboxylic acid, the low bias conductances resulting from the four binding modes are all found to have either a high or a low value, well consistent with recent experimental observations. The trend can be rationalized by the behavior of electrode-induced gap states at small bias. When bias increases to higher values, states from the anchoring groups enter into the bias window and contribute significantly to the tunneling process so that transport properties become more complicated for the four binding modes. Other low bias behaviors including the values of the inverse length scale for tunneling characteristic, contact resistance, and the ratios of the high/low conductance values are also calculated and compared to experimental results. The conducting capabilities of the three anchoring groups are found to decrease from dithiol, diamine to dicarboxylic-acid, largely owing to a decrease in binding strength to the electrodes. Our results give a clear microscopic picture to the transport physics and provide reasonable qualitative explanations for the corresponding experimental data.

The Social Sensitivity of Preschoolers in Peer Conflicts: Do Children Act Differently with Different Peers?

ERIC Educational Resources Information Center

Rourke, Mary T.; Wozniak, Robert H.; Cassidy, Kimberly Wright

1999-01-01

Examined how aspects of preschoolers' peer-conflict behavior varied according to partner. Found that initiating and negotiating behavior in early sessions better predicted behavior in the fourth session for the same-partner versus change-partner group. Results indicated that much of preschoolers' conflict behavior was sensitive to influences…

Effects of ayahuasca on the development of ethanol-induced behavioral sensitization and on a post-sensitization treatment in mice.

PubMed

Oliveira-Lima, A J; Santos, R; Hollais, A W; Gerardi-Junior, C A; Baldaia, M A; Wuo-Silva, R; Yokoyama, T S; Costa, J L; Malpezzi-Marinho, E L A; Ribeiro-Barbosa, P C; Berro, L F; Frussa-Filho, R; Marinho, E A V

2015-04-01

Hallucinogenic drugs were used to treat alcoholic patients in the past, and recent developments in the study of hallucinogens led to a renewal of interest regarding the application of these drugs in the treatment of addiction. In this scenario, accumulating evidence suggests that the hallucinogenic brew ayahuasca (Aya) may have therapeutic effects on substance abuse problems. We investigated the effects of Aya on spontaneous locomotor activity and ethanol(Eth)-induced hyperlocomotion and subsequent locomotor sensitization by a two-injection protocol. Additionally, we tested the effect of Aya on an 8-day counter-sensitization protocol to modify sensitized responses induced by a repeated treatment with Eth (1.8g/kg) for 8 alternate days. Aya showed high sensitivity in preventing the development of Eth-induced behavioral sensitization, attenuating it at all doses (30, 100, 200, 300 or 500 mg/kg) without modifying spontaneous locomotor activity. At the highest doses (300 and 500 mg/kg), Aya also showed selectivity to both acute and sensitized Eth responses. Finally, a counter-sensitization strategy with 100 or 300 mg/kg of Aya for 8 consecutive days after the establishment of Eth-induced behavioral sensitization was effective in blocking its subsequent expression on an Eth challenge. We demonstrated that Aya not only inhibits early behaviors associated with the initiation and development of Eth addiction, but also showed effectiveness in reversing long-term drug effects expression, inhibiting the reinstatement of Eth-induced behavioral sensitization when administered in the Eth-associated environment. Copyright © 2015 Elsevier Inc. All rights reserved.

Alpha1-adrenergic drugs affect the development and expression of ethanol-induced behavioral sensitization.

PubMed

Kim, Andrezza Kyunmi; Souza-Formigoni, Maria Lucia Oliveira

2013-11-01

According to the incentive sensitization theory, addiction is caused primarily by drug-induced sensitization in the brain mesocorticolimbic systems. After repeated ethanol administration, some animals develop psychomotor sensitization, a phenomenon which occurs simultaneously with the incentive sensitization. Recent evidence suggests the involvement of norepinephrine (NE) in drug addiction, with a critical role in the ethanol reinforcing properties. In this study we evaluated the influence of an agonist (phenylephrine) and an antagonist (prazosin) of alpha1-adrenergic receptors on the development and expression of behavioral sensitization to ethanol. Male Swiss mice, previously treated with ethanol or saline, were challenged with the combined administration of ethanol (or saline) with alpha1-adrenergic drugs. Prazosin (0.1; 0.5 and 1.0 mg/kg) and phenylephrine (1.0 and 2.0 mg/kg) administration blocked the expression of behavioral sensitization to ethanol. In another set of experiments, mice treated with 0.5mg/kg of prazosin+ethanol did not present the development of behavioral sensitization. However, when challenged with ethanol alone, they showed the same sensitized levels of locomotor activity of those presented by mice previously treated with ethanol and saline. Phenylephrine (1.0 mg/kg) treatment did not affect the development of behavioral sensitization. Based on this data, we concluded that the alteration of alpha1-adrenergic receptors functioning, by the administration agonists or antagonists, affected the locomotor sensitization to the stimulant effect of ethanol, suggesting that the normal functioning of the noradrenergic system is essential to its development and expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Behavioral cross-sensitization between DOCA-induced sodium appetite and cocaine-induced locomotor behavior

PubMed Central

Acerbo, Martin J.; Johnson, Alan Kim

2011-01-01

Behavioral sensitization involves increases in the magnitude of a response to a stimulus after repeated exposures to the same response initiator. Administration of psychomotor stimulants and the induction of appetitive motivational states associated with natural reinforcers like sugar and salt are among experimental manipulations producing behavioral sensitization. In rats, repeated administration of the mineralocorticoid agonist deoxycorticosterone acetate (DOCA) initially induces incremental increases in daily hypertonic saline consumption (i.e., sensitization of sodium appetite) in spite of the retention of sodium. The present studies investigated whether sodium appetite sensitization induced by DOCA shares mechanisms similar to those of psychomotor stimulant-induced sensitization, and whether there is evidence for reciprocal cross-sensitization. In Experiments 1 and 3, rats received control or cocaine treatments to induce locomotor sensitization. A week later DOCA (or vehicle) was administered to generate a sodium appetite. Animals pretreated with cocaine showed a greater sodium appetite. In Experiment 2, the order of the putative sensitizing treatments was reversed. Rats first received either a series of DOCA or vehicle treatments either with or without access to saline and were later tested for sensitization of the locomotor response to cocaine. Animals pretreated with DOCA without access to saline showed greater locomotor responses to cocaine than animals receiving vehicle treatments. Together these experiments indicate that treatments generating a sustained salt appetite and producing cocaine-induced psychomotor responses show reciprocal behavioral cross-sensitization. The underlying mechanisms accounting for this relationship may be the fact that psychostimulants and an unresolved craving for sodium can act as potent stressors. PMID:21352848

Bilateral six-hydroxydopamine administration to PFC prevents the expression of behavioral sensitization to methylphenidate.

PubMed

Wanchoo, S J; Lee, M J; Swann, A C; Dafny, N

2010-02-02

Psychostimulants like amphetamine and methylphenidate (MPD) are used to treat attention deficit hyperactivity disorder (ADHD), which is marked by developmentally inappropriate inattention, hyperactivity, and impulsivity. Neuropsychological analyses indicate that ADHD patients are impaired on tasks of behavioral inhibition, reward reversal, and working memory, which are functions of the prefrontal cortex (PFC) and are modulated by the mesocortical dopamine (DA) system. Non-specific electrical lesioning of PFC eliminated the expression of behavioral sensitization elicited by chronic MPD administration. Behavioral sensitization is the progressive augmentation of locomotor activity as a result of repetitive (chronic) exposure to the drug. It is believed that the sensitization to chronic drug treatment is caused due to an increase in DA in the mesocorticolimbic DA system, which includes the PFC. Therefore, this study investigated the role of PFC DA in mediating the behavioral sensitization to repeated administration of MPD in adult male Sprague-Dawley rats. On experimental day (ED) 1, the behavior was recorded post-saline injection. On ED 2, the rats were divided into three groups--control, sham and bilateral 6-OHDA treated group; and the sham and 6-OHDA treated groups underwent respective surgeries. After 5 days of rest following surgery, the post-surgery baseline was recorded on ED 8 following a saline injection. All three groups received 2.5 mg/kg MPD for 6 days (from ED 9 to ED 14), followed by a 3-day washout period (ED 15 to ED 18). On ED 19, a rechallenge injection of 2.5 mg/kg MPD was given and locomotor activity was recorded. It was found that the 6-OHDA lesion group failed to exhibit behavioral sensitization to MPD. The involvement of the dopaminergic afferents of PFC in behavioral sensitization to MPD is discussed. (c) 2009 Elsevier B.V. All rights reserved.

Selective labelling of diazepam-insensitive GABAA receptors in vivo using [3H]Ro 15-4513.

PubMed

Pym, Luanda J; Cook, Susan M; Rosahl, Thomas; McKernan, Ruth M; Atack, John R

2005-11-01

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.

The Detection of Protein via ZnO Resonant Raman Scattering Signal

NASA Astrophysics Data System (ADS)

Shan, Guiye; Yang, Guoliang; Wang, Shuang; Liu, Yichun

2008-03-01

Detecting protein with high sensitivity and specificity is essential for disease diagnostics, drug screening and other application. Semiconductor nanoparticles show better properties than organic dye molecules when used as markers for optical measurements. We used ZnO nanoparticles as markers for detecting protein in resonant Raman scattering measurements. The highly sensitive detection of proteins was achieved by an antibody-based sandwich assay. A probe for the target protein was constructed by binding the ZnO/Au nanoparticles to a primary antibody by eletrostatic interaction between Au and the antibody. A secondary antibody, which could be specifically recognized by target protein, was attached to a solid surface. The ZnO/Au-antibody probe could specifically recognize and bind to the complex of the target protein and secondary antibody. Our measurements using the resonant Raman scattering signal of ZnO nanoparticles showed good selectivity and sensitivity for the target protein.

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors

PubMed Central

2018-01-01

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity. PMID:29768011

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors.

PubMed

Gao, Zhaoli; Xia, Han; Zauberman, Jonathan; Tomaiuolo, Maurizio; Ping, Jinglei; Zhang, Qicheng; Ducos, Pedro; Ye, Huacheng; Wang, Sheng; Yang, Xinping; Lubna, Fahmida; Luo, Zhengtang; Ren, Li; Johnson, Alan T Charlie

2018-06-13

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

PubMed

Furutani, Shogo; Okuhara, Daiki; Hashimoto, Anju; Ihara, Makoto; Kai, Kenji; Hayashi, Hideo; Sattelle, David B; Matsuda, Kazuhiko

2017-10-01

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Phenanthrene binding by humic acid-protein complexes as studied by passive dosing technique.

PubMed

Zhao, Jian; Wang, Zhenyu; Ghosh, Saikat; Xing, Baoshan

2014-01-01

This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Copyright © 2013 Elsevier Ltd. All rights reserved.

Distinguishing between learning and motivation in behavioral tests of the reinforcement sensitivity theory of personality.

PubMed

Smillie, Luke D; Dalgleish, Len I; Jackson, Chris J

2007-04-01

According to Gray's (1973) Reinforcement Sensitivity Theory (RST), a Behavioral Inhibition System (BIS) and a Behavioral Activation System (BAS) mediate effects of goal conflict and reward on behavior. BIS functioning has been linked with individual differences in trait anxiety and BAS functioning with individual differences in trait impulsivity. In this article, it is argued that behavioral outputs of the BIS and BAS can be distinguished in terms of learning and motivation processes and that these can be operationalized using the Signal Detection Theory measures of response-sensitivity and response-bias. In Experiment 1, two measures of BIS-reactivity predicted increased response-sensitivity under goal conflict, whereas one measure of BAS-reactivity predicted increased response-sensitivity under reward. In Experiment 2, two measures of BIS-reactivity predicted response-bias under goal conflict, whereas a measure of BAS-reactivity predicted motivation response-bias under reward. In both experiments, impulsivity measures did not predict criteria for BAS-reactivity as traditionally predicted by RST.

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase.

PubMed

Nagatoishi, Satoru; Yamaguchi, Sou; Katoh, Etsuko; Kajita, Keita; Yokotagawa, Takane; Kanai, Satoru; Furuya, Toshio; Tsumoto, Kouhei

2018-05-01

19 F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19 F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19 F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19 F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments. Copyright © 2018 Elsevier Ltd. All rights reserved.

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

PubMed

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

PubMed

Mazzochi, Christopher; Bubien, James K; Smith, Peter R; Benos, Dale J

2006-03-10

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

NASA Astrophysics Data System (ADS)

Geer, M. Ariel; Fitzgerald, Michael C.

2016-02-01

The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270

PubMed Central

Navarro, Claudio A.; von Bernath, Diego; Martínez-Bussenius, Cristóbal; Castillo, Rodrigo A.

2015-01-01

Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium. PMID:26637599

Anticancer drug-DNA interactions measured using a photoinduced electron-transfer mechanism based on luminescent quantum dots.

PubMed

Yuan, Jipei; Guo, Weiwei; Yang, Xiurong; Wang, Erkang

2009-01-01

A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L(-1) and a linear detection range from 10 nmol L(-1) to 4.5 micromol L(-1) was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

Women's finger sensitivity correlates with partnered sexual behavior but not solitary masturbation frequencies.

PubMed

Brody, Stuart; Fischer, Agneta H; Hess, Ursula

2008-01-01

In a sample of 97 healthy Dutch female university students, women with greater finger tactile sensitivity (von Frey-type filaments) engaged more in partnered (but not solitary masturbation) sexual behavior. Orgasmic responses in the past 30 days were not correlated with finger sensitivity. Results are discussed in terms of differences between different sexual behaviors, as well as susceptibility to reinforcement, and psychoanalytic views of conversion hysteria.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods.

PubMed

Schuitemaker, Alie; van Berckel, Bart N M; Kropholler, Marc A; Veltman, Dick J; Scheltens, Philip; Jonker, Cees; Lammertsma, Adriaan A; Boellaard, Ronald

2007-05-01

(R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).

Novel regulation of Skp1 by the Dictyostelium AgtA α-galactosyltransferase involves the Skp1-binding activity of its WD40 repeat domain.

PubMed

Schafer, Christopher M; Sheikh, M Osman; Zhang, Dongmei; West, Christopher M

2014-03-28

The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function.

Executive function, approach sensitivity, and emotional decision making as influences on risk behaviors in young adults.

PubMed

Patrick, Megan E; Blair, Clancy; Maggs, Jennifer L

2008-05-01

Relations among executive function, behavioral approach sensitivity, emotional decision making, and risk behaviors (alcohol use, drug use, and delinquent behavior) were examined in single female college students (N = 72). Hierarchical multiple regressions indicated a significant Approach Sensitivity x Working Memory interaction in which higher levels of alcohol use were associated with the combination of greater approach tendency and better working memory. This Approach Sensitivity x Working Memory interaction was also marginally significant for drug use and delinquency. Poor emotional decision making, as measured by a gambling task, was also associated with higher levels of alcohol use, but only for individuals low in inhibitory control. Findings point to the complexity of relations among aspects of self-regulation and personality and provide much needed data on neuropsychological correlates of risk behaviors in a nonclinical population.

[Study on the aggregation behavior of cationic porphyrins and their interaction with ctDNA].

PubMed

Ma, Hong-Min; Chen, Xin; Sun, Shu-Ting; Zhang, Li-Na; Wu, Dan; Zhu, Pei-Hua; Li, Yan; Du, Bin; Wei, Qin

2009-02-01

Interest in the interaction between cationic porphyrins, particularly derivatives of meso-tetra(N-methylpyridinium-4-yl) porphyrin(TMPyP), and DNA abounds because they are versatile DNA-binding agents that could find application in photodynamic therapy, cancer detection, artificial nucleases, virus inhibition and so on. The interaction of two water-soluble cationic porphyrins, meso-tetrakis(4-N, N, N-trimethylanilinium) porphyrin (TMAP) and 5-phenyl-10,15,20-tris[4-(N-methyl) pyridinium]porphyrin (TriMPyP), with calf thymus DNA (ctDNA) was studied by UV-Vis absorption spectroscopy, fluorescence spectroscopy and resonance light scattering technique. TriMPyP forms aggregate in water due to the molecular asymmetry while TMAP exists as monomers. At lower concentrations of ctDNA (R > 1, R = c(TMAP)/c(DNA) base pair), the interaction of TMAP with DNA leads to significant hypochromicity and bathochromic shift of absorption spectra. And the fluorescence of TMAP was quenched while it showed enhanced resonance light scattering signals. But the extent of enhancement of resonance light scattering signals is very small, so the aggregate of TMAP is not very high. These observations indicate the self-stacking of TMAP along the DNA surface. At higher concentrations of ctDNA (R < 1), TMAP association with DNA is via outside binding which is accompanied with hyperchromic effect and fluorescence enhancement while the resonance light scattering signals is reduced. DNA addition decreases the fluorescence intensity of TriMPyP and it shifts the peak to the higher wavelengths (red shift). The interaction with DNA promotes the aggregation of TriMPyP and no simple outside binding is observed even at higher concentrations of ctDNA. The steric effect of molecular distortion constrains the intercalation or further binding to DNA. The effect of ionic strength on the interaction was investigated at two DNA concentrations, 1.2 and 24.0 micromol x L(-1), for TMAP. The Interactions of both porphyrins with DNA show high sensitivity to ionic strength. By addition of NaCl, electrostatic attraction is decreased, resulting in the change of binding mode.

Naproxen Attenuates Sensitization of Depressive-Like Behavior and Fever during Maternal Separation

PubMed Central

Hennessy, Michael B.; Stafford, Nathan P.; Yusko-Osborne, Brittany; Schiml, Patricia A.; Xanthos, Evan D.; Deak, Terrence

2014-01-01

Early life stress can increase susceptibility for later development of depressive illness though a process thought to involve inflammatory mediators. Isolated guinea pig pups exhibit a passive, depressive-like behavioral response and fever that appear mediated by proinflammatory activity, and which sensitize with repeated separations. Treatment with an anti-inflammatory can attenuate the behavioral response during the initial separation and separation the following day. Here we used the cyclooxygenase inhibitor naproxen to examine the role of prostaglandins in mediating the depressive-like behavior and core body temperature of young guinea pigs during an initial separation, separation the next day, and separation 10 days after the first. The passive, depressive-like behavior as well as fever sensitized with repeated separation. Three days of injection with 14 mg/kg of naproxen prior to the initial separation reduced depressive-like behavior during all three separations. A 28 mg/kg dose of naproxen, however, had minimal effect on behavior. Fever during the early separations was moderated by naproxen, but only at the higher dose. These results suggest a role of prostaglandins in the behavioral and febrile response to maternal separation, and particularly in the sensitization of depressive-like behavior following repeated separation. PMID:25449392

Altered interregional molecular associations of the serotonin transporter in attention deficit/hyperactivity disorder assessed with PET.

PubMed

Vanicek, Thomas; Kutzelnigg, Alexandra; Philippe, Cecile; Sigurdardottir, Helen L; James, Gregory M; Hahn, Andreas; Kranz, Georg S; Höflich, Anna; Kautzky, Alexander; Traub-Weidinger, Tatjana; Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus; Kasper, Siegfried; Lanzenberger, Rupert

2017-02-01

Altered serotonergic neurotransmission has been found to cause impulsive and aggressive behavior, as well as increased motor activity, all exemplifying key symptoms of ADHD. The main objectives of this positron emission tomography (PET) study were to investigate the serotonin transporter binding potential (SERT BP ND ) in patients with ADHD and to assess associations of SERT BP ND between the brain regions. 25 medication-free patients with ADHD (age ± SD; 32.39 ± 10.15; 10 females) without any psychiatric comorbidity and 25 age and sex matched healthy control subjects (33.74 ± 10.20) were measured once with PET and the highly selective and specific radioligand [ 11 C]DASB. SERT BP ND maps in nine a priori defined ROIs exhibiting high SERT binding were compared between groups by means of a linear mixed model. Finally, adopted from structural and functional connectivity analyses, we performed correlational analyses using regional SERT binding potentials to examine molecular interregional associations between all selected ROIs. We observed significant differences in the interregional correlations between the precuneus and the hippocampus in patients with ADHD compared to healthy controls, using SERT BP ND of the investigated ROIs (P < 0.05; Bonferroni corrected). When correlating SERT BP ND and age in the ADHD and the healthy control group, we confirmed an age-related decline in brain SERT binding in the thalamus and insula (R 2  = 0.284, R 2  = 0.167, Ps < 0.05; Bonferroni corrected). The results show significantly different interregional molecular associations of the SERT expression for the precuneus with hippocampus in patients with ADHD, indicating presumably altered functional coupling. Altered interregional coupling between brain regions might be a sensitive approach to demonstrate functional and molecular alterations in psychiatric conditions. Hum Brain Mapp 38:792-802, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Binding-Induced Fluorescence of Serotonin Transporter Ligands: A Spectroscopic and Structural Study of 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) and APP+ Analogues

PubMed Central

2014-01-01

The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP+) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP+) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP+), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

Interaction of growth hormone receptor/binding protein gene disruption and caloric restriction for insulin sensitivity and attenuated aging

PubMed Central

Arum, Oge; Saleh, Jamal; Boparai, Ravneet; Turner, Jeremy; Kopchick, John; Khardori, Romesh; Bartke, Andrzej

2014-01-01

The correlation of physiological sensitivity to insulin ( vis-à-vis glycemic regulation) and longevity is extensively established, creating a justifiable gerontological interest on whether insulin sensitivity is causative, or even predictive, of some or all phenotypes of slowed senescence (including longevity). The growth hormone receptor/ binding protein gene-disrupted (GHR-KO) mouse is the most extensively investigated insulin-sensitive, attenuated aging model. It was reported that, in a manner divergent from similar mutants, GHR-KO mice fail to respond to caloric restriction (CR) by altering their insulin sensitivity. We hypothesized that maximized insulin responsiveness is what causes GHR-KO mice to exhibit a suppressed survivorship response to dietary (including caloric) restriction; and attempted to refute this hypothesis by assessing the effects of CR on GHR-KO mice for varied slow-aging-associated phenotypes. In contrast to previous reports, we found GHR-KO mice on CR to be less responsive than their ad libitum (A.L.) counterparts to the hypoglycemia-inducing effects of insulin. Further, CR had negligible effects on the metabolism or cognition of GHR-KO mice. Therefore, our data suggest that the effects of CR on the insulin sensitivity of GHR-KO mice do not concur with the effects of CR on the aging of GHR-KO mice. PMID:25789159

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Protein Surface Structural Recognition in Inactive Areas: A New Immobilization Strategy for Acetylcholinesterase.

PubMed

Diao, Jianxiong; Yu, Xiaolu; Ma, Lin; Li, Yuanqing; Sun, Ying

2018-05-16

This work reported a new method of design for the immobilization of acetylcholinesterase (AChE) based on its molecular structure to improve its sensitivity and stability. The immobilization binding site on the surface of AChE was determined using MOLCAD's multi-channel functionality. Then, 11 molecules ((+)-catechin, (-)-epicatechin, (-)-gallocatechin, hesperetin, naringenin, quercetin, taxifolin, (-)-epicatechin gallate, flupirtine, atropine, and hyoscyamine) were selected from the ZINC database (about 50 000 molecules) as candidate affinity ligands for AChE. The fluorescence results showed that the binding constant K b between AChE and the ligands ranged from 0.01344 × 10 4 to 4.689 × 10 4 M -1 and there was one independent class of binding site for the ligands on AChE. The AChE-ligand binding free energy ranged from -12.14 to -26.65 kJ mol -1 . Naringenin, hesperetin, and quercetin were the three most potent immobilized affinity ligands. In addition, it was confirmed that the binding between the immobilized ligands only occurred at a single site, located in an inactive area on the surface of AChE, and did not affect the enzymatic activity as shown through a competition experiment and enzyme assay. This method based on protein surface structural recognition with high sensitivity and stability can be used as a generic approach for design of the enzyme immobilization and biosensor development.

Quantification of [11C]yohimbine binding to α2 adrenoceptors in rat brain in vivo

PubMed Central

Phan, Jenny-Ann; Landau, Anne M; Wong, Dean F; Jakobsen, Steen; Nahimi, Adjmal; Doudet, Doris J; Gjedde, Albert

2015-01-01

We quantified the binding potentials (BPND) of [11C]yohimbine binding in rat brain to alpha-2 adrenoceptors to evaluate [11C]yohimbine as an in vivo marker of noradrenergic neurotransmission and to examine its sensitivity to the level of noradrenaline. Dual [11C]yohimbine dynamic positron emission tomography (PET) recordings were applied to five Sprague Dawley rats at baseline, followed by acute amphetamine administration (2 mg/kg) to induce elevation of the endogenous level of noradrenaline. The volume of distribution (VT) of [11C]yohimbine was obtained using Logan plot with arterial plasma input. Because alpha-2 adrenoceptors are distributed throughout the brain, the estimation of the BPND is complicated by the absence of an anatomic region of no displaceable binding. We used the Inhibition plot to acquire the reference volume, VND, from which we calculated the BPND. Acute pharmacological challenge with amphetamine induced a significant decline of [11C]yohimbine BPND of ~38% in all volumes of interest. The BPND was greatest in the thalamus and striatum, followed in descending order by, frontal cortex, pons, and cerebellum. The experimental data demonstrate that [11C]yohimbine binding is sensitive to a challenge known to increase the extracellular level of noradrenaline, which can benefit future PET investigations of pathologic conditions related to disrupted noradrenergic neurotransmission. PMID:25564241

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021