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Sample records for specific alcohol binding

  1. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  2. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum. Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols.

    Science.gov (United States)

    Fraaije, M W; Veeger, C; van Berkel, W J

    1995-11-15

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycols and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred. Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the C gamma atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation. The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa < 6, 25 degrees C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcohol-oxidase-catalyzed conversion of 4-allylphenols.

  3. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  4. Parental alcohol use, alcohol-related problems, and alcohol-specific attitudes, alcohol-specific communication, and adolescent excessive alcohol use and alcohol-related problems: An indirect path model

    NARCIS (Netherlands)

    Mares, S.H.W.; Vorst, H. van der; Engels, R.C.M.E.; Lichtwarck-Aschoff, A.

    2011-01-01

    Alcohol-specific parent-child communication has often been studied in relation to regular alcohol use of adolescents. However, it might be as important to focus on adolescent problematic alcohol use. In addition, the way parents communicate with their children about alcohol might depend on their own

  5. Ethanol intake and 3H-serotonin uptake II: A study in alcoholic patients using platelets 3H-paroxetine binding

    International Nuclear Information System (INIS)

    Daoust, M.; Boucly, P.; Ernouf, D.; Breton, P.; Lhuintre, J.P.

    1991-01-01

    The kinetic parameters of 3 H-paroxetine binding and 3 H-serotonin uptake were studied in platelets of alcoholic patients. There was no difference between alcoholic and non alcoholic subjects in 3 H-paroxetine binding. When binding and 3 H-serotonin uptake were studied, in the same plasma of the same subjects, the Vmax of serotonin uptake was increased in alcoholics. The data confirm the involvement of serotonin uptake system in alcohol dependance and suggest that serotonin uptake and paroxetine binding sites may be regulated independently in this pathology

  6. Alcohol-specific parenting, adolescent alcohol use and the mediating effect of adolescent alcohol-related cognitions

    NARCIS (Netherlands)

    Mares, S.H.W.; Lichtwarck-Aschoff, A.; Engels, R.C.M.E.

    2013-01-01

    Objectives : Previous research indicated that alcohol-specific parenting is an important precursor of adolescent alcohol use, but failed to define the underlying mechanism. Based on social cognitive theory, alcohol-related cognitions such as alcohol refusal self-efficacy and alcohol-related

  7. Alcohol binding in the C1 (C1A + C1B) domain of protein kinase C epsilon

    Science.gov (United States)

    Pany, Satyabrata; Das, Joydip

    2015-01-01

    Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε. Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists. PMID:26210390

  8. The role of parental alcohol-specific communication in early adolescents’ alcohol use

    NARCIS (Netherlands)

    Vorst, H. van der; Burk, W.J.; Engels, R.C.M.E.

    2010-01-01

    Many alcohol prevention programs advocate conversations about alcohol between parents and children because verbal communication is the most direct way for parents to express their thoughts, rules, and concerns about alcohol to their children, so called alcohol-specific communication. Nevertheless,

  9. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism

    International Nuclear Information System (INIS)

    Noble, E.P.; Blum, K.; Ritchie, T.; Montgomery, A.; Sheridan, P.J.

    1991-01-01

    The allelic association of the human D2 dopamine receptor gene with the binding characteristics of the D2 dopamine receptor was determined in 66 brains of alcoholic and non-alcoholic subjects. In a blinded experiment, DNA from the cerebral cortex was treated with the restriction endonuclease Taql and probed with a 1.5-kilobase (kb) digest of a clone (lambda hD2G1) of the human D2 dopamine receptor gene. The binding characteristics (Kd [binding affinity] and Bmax [number of binding sites]) of the D2 dopamine receptor were determined in the caudate nuclei of these brains using tritiated spiperone as the ligand. The adjusted Kd was significantly lower in alcoholic than in nonalcoholic subjects. In subjects with the A1 allele, in whom a high association with alcoholism was found, the Bmax was significantly reduced compared with the Bmax of subjects with the A2 allele. Moreover, a progressively reduced Bmax was found in subjects with A2/A2, A1/A2, and A1/A1 alleles, with subjects with A2/A2 having the highest mean values, and subjects with A1/A1, the lowest. The polymorphic pattern of the D2 dopamine receptor gene and its differential expression of receptors suggests the involvement of the dopaminergic system in conferring susceptibility to at least one subtype of severe alcoholism

  10. Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference.

    Science.gov (United States)

    Hwang, B.H.; Kunkler, P.E.; Lumeng, L.

    1997-01-01

    It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In

  11. Influence of alcoholism and cholesterol on TSPO binding in brain: PET [11C]PBR28 studies in humans and rodents.

    Science.gov (United States)

    Kim, Sung Won; Wiers, Corinde E; Tyler, Ryan; Shokri-Kojori, Ehsan; Jang, Yeon Joo; Zehra, Amna; Freeman, Clara; Ramirez, Veronica; Lindgren, Elsa; Miller, Gregg; Cabrera, Elizabeth A; Stodden, Tyler; Guo, Min; Demiral, Şükrü B; Diazgranados, Nancy; Park, Luke; Liow, Jeih-San; Pike, Victor; Morse, Cheryl; Vendruscolo, Leandro F; Innis, Robert B; Koob, George F; Tomasi, Dardo; Wang, Gene-Jack; Volkow, Nora D

    2018-05-03

    Neuroinflammation appears to contribute to neurotoxicity observed with heavy alcohol consumption. To assess whether chronic alcohol results in neuroinflammation we used PET and [ 11 C]PBR28, a ligand that binds to the 18-kDa translocator protein (TSPO), to compare participants with an alcohol use disorder (AUD: n = 19) with healthy controls (HC: n = 17), and alcohol-dependent (n = 9) with -nondependent rats (n = 10). Because TSPO is implicated in cholesterol's transport for steroidogenesis, we investigated whether plasma cholesterol levels influenced [ 11 C]PBR28 binding. [ 11 C]PBR28 binding did not differ between AUD and HC. However, when separating by TSPO genotype rs6971, we showed that medium-affinity binders AUD participants showed lower [ 11 C]PBR28 binding than HC in regions of interest (whole brain, gray and white matter, hippocampus, and thalamus), but no group differences were observed in high-affinity binders. Cholesterol levels inversely correlated with brain [ 11 C]PBR28 binding in combined groups, due to a correlation in AUD participants. In rodents, we observed no differences in brain [ 11 C]PBR28 uptake between alcohol-dependent and -nondependent rats. These findings, which are consistent with two previous [ 11 C]PBR28 PET studies, may indicate lower activation of microglia in AUD, whereas failure to observe alcohol effects in the rodent model indicate that species differences do not explain the discrepancy with prior rodent autoradiographic studies reporting increases in TSPO binding with chronic alcohol. However, reduced binding in AUD participants could also reflect competition from endogenous TSPO ligands such as cholesterol; and since the rs6971 polymorphism affects the cholesterol-binding domain of TSPO this could explain why differences were observed only in medium-affinity binders.

  12. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit......Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length...... than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has...

  13. Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use

    DEFF Research Database (Denmark)

    Erritzoe, D.; Frokjaer, V. G.; Haugbol, S.

    2009-01-01

    receptor (5-HT(2A)) in humans, we tested in 136 healthy human subjects if body mass index (BMI), degree of alcohol consumption and tobacco smoking was associated to the cerebral in vivo 5-HT(2A) receptor binding as measured with (18)F-altanserin PET. The subjects' BMI's ranged from 18.4 to 42.8 (25.......2+/-4.3) kg/m(2). Cerebral cortex 5-HT(2A) binding was significantly positively correlated to BMI, whereas no association between cortical 5-HT(2A) receptor binding and alcohol or tobacco use was detected. We suggest that our observation is driven by a lower central 5-HT level in overweight people, leading...

  14. Dynamic Multi-Component Covalent Assembly for the Reversible Binding of Secondary Alcohols and Chirality Sensing

    Science.gov (United States)

    You, Lei; Berman, Jeffrey S.; Anslyn, Eric V.

    2011-01-01

    Reversible covalent bonding is often employed for the creation of novel supramolecular structures, multi-component assemblies, and sensing ensembles. In spite of remarkable success of dynamic covalent systems, the reversible binding of a mono-alcohol with high strength is challenging. Here we show that a strategy of carbonyl activation and hemiaminal ether stabilization can be embodied in a four-component reversible assembly that creates a tetradentate ligand and incorporates secondary alcohols with exceptionally high affinity. Evidence is presented that the intermediate leading to binding and exchange of alcohols is an iminium ion. Further, to demonstrate the use of this assembly process we explored chirality sensing and enantiomeric excess determinations. An induced twist in the ligand by a chiral mono-ol results in large Cotton effects in the circular dichroism spectra indicative of the alcohol’s handedness. The strategy revealed in this study should prove broadly applicable for the incorporation of alcohols into supramolecular architecture construction. PMID:22109274

  15. Alcohol-specific parenting, adolescents' self-control, and alcohol use: a moderated mediation model.

    Science.gov (United States)

    Koning, Ina M; van den Eijnden, Regina J J M; Vollebergh, Wilma A M

    2014-01-01

    There is convincing evidence that parental rules about alcohol are important in curbing adolescents' alcohol use. However, little is known about the mechanisms through which the direct link between alcohol-specific parenting and alcohol use is obtained. In this study, we investigated the mediating effect of adolescent self-control on the relationship between alcohol-specific rules and adolescents' drinking behavior and whether this mediation effect depends on the level of quality of communication. A total of 883 adolescents participated in this longitudinal study at ages 13, 14, and 15 years. Strict rules predicted lower rates of drinking, but no direct effect of the quality of communication on adolescents' alcohol use was found. A higher level of self-control was related to lower rates of drinking in adolescents. The indirect effect of rules about alcohol through adolescents' self-control was statistically significant, yet only in adolescents with high qualitative parent-child communication about alcohol. In adolescents with low quality of parent-child communication, self-control was not related to drinking. These findings imply that strict rule setting in combination with qualitative parent-child communication is an important target for prevention. In addition, findings point at the importance of high qualitative parent-child communication for adolescents' motivation to engage in self-control to avoid drinking.

  16. 78 FR 26849 - Model Specifications for Breath Alcohol Ignition Interlock Devices (BAIIDs)

    Science.gov (United States)

    2013-05-08

    ... requirements, and asked whether the Model Specifications should limit sensor technology to alcohol-specific sensors (such as fuel cell technology based on electrochemical oxidation of alcohol) or other emerging... have demanded alcohol- specific sensor technology. [Interlocks that] are not alcohol-specific...

  17. Polyvinyl-alcohol-based magnetic beads for rapid and efficient separation of specific or unspecific nucleic acid sequences

    International Nuclear Information System (INIS)

    Oster, J.; Parker, Jeffrey; Brassard, Lothar

    2001-01-01

    The versatile application of polyvinyl-alcohol-based magnetic M-PVA beads is demonstrated in the separation of genomic DNA, sequence specific nucleic acid purification, and binding of bacteria for subsequent DNA extraction and detection. It is shown that nucleic acids can be obtained in high yield and purity using M-PVA beads, making sample preparation efficient, fast and highly adaptable for automation processes

  18. Activation of prefrontal cortex and anterior thalamus in alcoholic subjects on exposure to alcohol-specific cues.

    Science.gov (United States)

    George, M S; Anton, R F; Bloomer, C; Teneback, C; Drobes, D J; Lorberbaum, J P; Nahas, Z; Vincent, D J

    2001-04-01

    Functional imaging studies have recently demonstrated that specific brain regions become active in cocaine addicts when they are exposed to cocaine stimuli. To test whether there are regional brain activity differences during alcohol cue exposure between alcoholic subjects and social drinkers, we designed a functional magnetic resonance imaging (fMRI) protocol involving alcohol-specific cues. Ten non-treatment-seeking adult alcoholic subjects (2 women) (mean [SD] age, 29.9 [9.9] years) as well as 10 healthy social drinking controls of similar age (2 women) (mean [SD] age, 29.4 [8.9] years) were recruited, screened, and scanned. In the 1.5-T magnetic resonance imaging scanner, subjects were serially rated for alcohol craving before and after a sip of alcohol, and after a 9-minute randomized presentation of pictures of alcoholic beverages, control nonalcoholic beverages, and 2 different visual control tasks. During picture presentation, changes in regional brain activity were measured with the blood oxygen level-dependent technique. Alcoholic subjects, compared with the social drinking subjects, reported higher overall craving ratings for alcohol. After a sip of alcohol, while viewing alcohol cues compared with viewing other beverage cues, only the alcoholic subjects had increased activity in the left dorsolateral prefrontal cortex and the anterior thalamus. The social drinkers exhibited specific activation only while viewing the control beverage pictures. When exposed to alcohol cues, alcoholic subjects have increased brain activity in the prefrontal cortex and anterior thalamus-brain regions associated with emotion regulation, attention, and appetitive behavior.

  19. The relationship between brand-specific alcohol advertising on television and brand-specific consumption among underage youth.

    Science.gov (United States)

    Ross, Craig S; Maple, Emily; Siegel, Michael; DeJong, William; Naimi, Timothy S; Ostroff, Joshua; Padon, Alisa A; Borzekowski, Dina L G; Jernigan, David H

    2014-08-01

    Being able to investigate the relationship between underage drinkers' preferences for particular brands and their exposure to advertising for those brands would represent a significant advance in alcohol marketing research. However, no previous national study has examined the relationship between underage youth exposure to brand-specific alcohol advertising and consumption of those brands. We conducted a cross-sectional, Internet-based survey of a national sample of 1,031 youth, ages 13-20, who had consumed at least 1 drink of alcohol in the past 30 days. We ascertained all alcohol brands consumed by respondents in the past 30 days. The main outcome measure was brand-specific consumption during the past 30 days, measured as a dichotomous variable. The main predictor variable was exposure to brand-specific alcohol advertising on television. The respondents reported which of 20 television shows popular with youth they had watched during the past 30 days. For each respondent, we calculated a standard measure of potential exposure to the brand-specific alcohol advertising that aired on those shows during the preceding 12 months, based on Nielsen (New York, NY) estimates of the youth audience for each show's telecasts. Compared to no brand-specific advertising exposure, any exposure was associated with an increased likelihood of brand-specific consumption (adjusted odds ratio 3.02; 95% confidence interval: 2.61-3.49) after controlling for several individual- and brand-level variables. When measured as a continuous variable, the relationship between advertising exposure and brand consumption was nonlinear, with a large association at lower levels of exposure and diminishing incremental effects as the level of exposure increased. There is a robust relationship between youth's brand-specific exposure to alcohol advertising on television and their consumption of those same alcohol brands during the past 30 days. This study provides further evidence of a strong

  20. Effects of general and alcohol-specific media literacy training on children's decision making about alcohol.

    Science.gov (United States)

    Austin, E W; Johnson, K K

    1997-01-01

    This article examines the immediate and delayed effects of media literacy training on third-grade children's perceptions of alcohol advertising, alcohol norms, expectancies for drinking, and behaviors toward alcohol. A Solomon four-group style experiment (N = 225) with two levels of the treatment factor assessed the effectiveness of in-school media literacy training for alcohol. The experiment compared a treatment that included the viewing of a videotape about television advertising along with the viewing of video clips of alcohol ads and discussion pertaining to alcohol advertising specifically versus one that included the viewing of the same general purpose media literacy videotape along with video clips of non-alcohol advertising and then discussion of advertising in general. The treatment had both immediate and delayed effects. Immediate effects included the children's increased understanding of persuasive intent, viewing of characters as less similar to people they knew in real life and less desirable, decreased desire to be like the characters, decreased expectation of positive consequences from drinking alcohol, and decreased likelihood to choose an alcohol-related product. Indirect effects also were found on their perceptions of television's realism and their views of social norms related to alcohol. Delayed effects were examined and confirmed on expectancies and behavior. The treatment was more effective when alcohol-specific, and it also was more effective among girls than boys.

  1. The Relationship between Brand-Specific Alcohol Advertising on Television and Brand-Specific Consumption among Underage Youth

    Science.gov (United States)

    Ross, Craig S.; Maple, Emily; Siegel, Michael; DeJong, William; Naimi, Timothy S.; Ostroff, Joshua; Padon, Alisa A.; Borzekowski, Dina L.G.; Jernigan, David H.

    2014-01-01

    Background Being able to investigate the relationship between underage drinkers' preferences for particular brands and their exposure to advertising for those brands would represent a significant advance in alcohol marketing research. However, no previous national study has examined the relationship between underage youth exposure to brand-specific alcohol advertising and consumption of those brands. Methods We conducted a cross-sectional, internet-based survey of a national sample of 1,031 youths, ages 13-20, who had consumed at least one drink of alcohol in the past 30 days. We ascertained all alcohol brands consumed by respondents in the past 30 days. The main outcome measure was brand-specific consumption during the past 30 days, measured as a dichotomous variable. The main predictor variable was exposure to brand-specific alcohol advertising on television. The respondents reported which of 20 television shows popular with youth they had watched during the past 30 days. For each respondent, we calculated a standard measure of potential exposure to the brand-specific alcohol advertising that aired on those shows during the preceding 12 months, based on Nielsen (New York, NY) estimates of the youth audience for each show's telecasts. Results Compared to no brand-specific advertising exposure, any exposure was associated with an increased likelihood of brand-specific consumption (adjusted odds ratio 3.02; 95% confidence interval: 2.61-3.49) after controlling for several individual- and brand-level variables. When measured as a continuous variable, the relationship between advertising exposure and brand consumption was nonlinear, with a large association at lower levels of exposure and diminishing incremental effects as the level of exposure increased. Conclusions There is a robust relationship between youth's brand-specific exposure to alcohol advertising on television and their consumption of those same alcohol brands during the past 30 days. This study provides

  2. Parental Alcohol-Specific Rules and Alcohol Use from Early Adolescence to Young Adulthood

    Science.gov (United States)

    Mares, Suzanne H. W.; Lichtwarck-Aschoff, Anna; Burk, William J.; van der Vorst, Haske; Engels, Rutger C. M. E.

    2012-01-01

    Background: Several studies stress the importance of alcohol-specific rules during adolescence to prevent them from drinking early and heavily. However, most studies have short follow-up periods and do not cover the relevant developmental period in which direct parental control diminishes and adolescent alcohol use increases. The current study…

  3. A comparison between brand-specific and traditional alcohol surveillance methods to assess underage drinkers' reported alcohol use.

    Science.gov (United States)

    Roberts, Sarah P; Siegel, Michael B; DeJong, William; Jernigan, David H

    2014-11-01

    Adolescent alcohol consumption remains common and is associated with many negative health outcomes. Unfortunately, common alcohol surveillance methods often underestimate consumption. Improved alcohol use measures are needed to characterize the landscape of youth drinking. We aimed to compare a standard quantity-frequency measure of youth alcohol consumption to a novel brand-specific measure. We recruited a sample of 1031 respondents across the United States to complete an online survey. Analyses included 833 male and female underage drinkers ages 13-20. Respondents reported on how many of the past 30 days they consumed alcohol, and the number of drinks consumed on an average drinking day. Using our brand-specific measure, respondents identified which brands they consumed, how many days they consumed each brand, and how many drinks per brand they usually had. Youth reported consuming significantly more alcohol (on average, 11 drinks more per month) when responding to the brand-specific versus the standard measure (p brands consumed (p brand preferences and consumption.

  4. 75 FR 61820 - Model Specifications for Breath Alcohol Ignition Interlock Devices (BAIIDs)

    Science.gov (United States)

    2010-10-06

    ... technology to alcohol-specific sensors (such as fuel cell technology based on electro-chemical oxidation of alcohol) or other emerging sensor technologies? Or, should NHTSA not specify the sensor technology and... require alcohol- specific technology in the Model Specifications, but that the particular sensor design...

  5. Alcohol marketing receptivity, marketing-specific cognitions, and underage binge drinking.

    Science.gov (United States)

    McClure, Auden C; Stoolmiller, Mike; Tanski, Susanne E; Engels, Rutger C M E; Sargent, James D

    2013-01-01

    Exposure to alcohol marketing is prevalent and is associated with both initiation and progression of alcohol use in underage youth. The mechanism of influence is not well understood, however. This study tests a model that proposes alcohol-specific cognitions as mediators of the relation between alcohol marketing and problematic drinking among experimental underage drinkers. This study describes a cross-sectional analysis of 1,734 U.S. 15- to 20-year-old underage drinkers, recruited for a national study of media and substance use. Subjects were queried about a number of alcohol marketing variables including TV time, Internet time, favorite alcohol ad, ownership of alcohol-branded merchandise (ABM), and exposure to alcohol brands in movies. The relation between these exposures and current (30-day) binge drinking was assessed, as were proposed mediators of this relation, including marketing-specific cognitions (drinker identity and favorite brand to drink), favorable alcohol expectancies, and alcohol norms. Paths were tested in a structural equation model that controlled for sociodemographics, personality, and peer drinking. Almost one-third of this sample of ever drinkers had engaged in 30-day binge drinking. Correlations between mediators were all statistically significant (range 0.16 to 0.47), and all were significantly associated with binge drinking. Statistically significant mediation was found for the association between ABM ownership and binge drinking through both drinker identity and having a favorite brand to drink, which also mediated the path between movie brand exposure and binge drinking. Peer drinking and sensation seeking were associated with binge drinking in paths through all mediators. Associations between alcohol marketing and binge drinking were mediated through marketing-specific cognitions that assess drinker identity and brand allegiance, cognitions that marketers aim to cultivate in the consumer. Copyright © 2012 by the Research Society on

  6. Saturation of retinol-binding protein correlates closely to the severity of alcohol-induced liver disease

    DEFF Research Database (Denmark)

    Wagnerberger, S.; Schäfer, C.; Bode, C.

    2006-01-01

    Impaired metabolism of retinol has been shown to occur in alcohol-induced liver disease (ALD). The purpose of the present study was to investigate the saturation of retinol-binding protein (RBP) in 6 patients with different stages of ALD. Hospitalized alcohol consumers (n=118) with different stages......: 43.5+/-6.2%; ALD3: 29.0+/-5.1%). The present study indicates that plasma concentrations of retinol and RBP per se do not correlate to severity of ALD, but rather that the retinol/RBP ratio links to the severity of alcohol-induced liver damage. From these results, a reduced availability of retinol...

  7. Alcohol Marketing Receptivity, Marketing-specific Cognitions and Underage Binge Drinking

    Science.gov (United States)

    McClure, Auden C.; Stoolmiller, Mike; Tanski, Susanne E.; Engels, Rutger C. M. E.; Sargent, James D.

    2012-01-01

    Background Exposure to alcohol marketing is prevalent and is associated with both initiation and progression of alcohol use in underage youth. The mechanism of influence is not well understood, however. This study tests a model that proposes alcohol-specific cognitions as mediators of the relation between alcohol marketing and problematic drinking among experimental underage drinkers. Methods This paper describes a cross-sectional analysis of 1734 U.S. 15–20 year old underage drinkers, recruited for a national study of media and substance use. Subjects were queried about a number of alcohol marketing variables including television time, internet time, favorite alcohol ad, ownership of alcohol branded merchandise (ABM), and exposure to alcohol brands in movies. The relation between these exposures and current (30 day) binge drinking was assessed, as were proposed mediators of this relation, including marketing-specific cognitions (drinker identity and favorite brand to drink), favorable alcohol expectancies and alcohol norms. Paths were tested in a structural equation model that controlled for socio-demographics, personality and peer drinking. Results Almost one-third of this sample of ever drinkers had engaged in 30 day binge drinking. Correlations among mediators were all statistically significant (range 0.16 – 0.47) and all were significantly associated with binge drinking. Statistically significant mediation was found for the association between ABM ownership and binge drinking through both drinker identity and having a favorite brand, which also mediated the path between movie brand exposure and binge drinking. Peer drinking and sensation seeking were associated with binge drinking in paths through all mediators. Conclusions Associations between alcohol marketing and binge drinking were mediated through marketing-specific cognitions that assess drinker identity and brand allegiance, cognitions that marketers aim to cultivate in the consumer. PMID:23256927

  8. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-01-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

  9. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Specific binding of atrial natriuretic factor in brain microvessels

    International Nuclear Information System (INIS)

    Chabrier, P.E.; Roubert, P.; Braquet, P.

    1987-01-01

    Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125 I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of 125 I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function

  11. Study of plasma binding of receptor-specific peptides

    OpenAIRE

    Gregor, David

    2008-01-01

    The binding ability of two receptor specific peptides namely 90Y-DOTA-TATE and 111In-DOTA-TATE was studied in therm of interspecies comparison by the method of equilibrium dialysis. This plasma protein binding was different for the chosen animal species (human, rat, rabbit, bovine eventually pork) whereas binding of 90Y-DOTA- TATE was higher than binding of 111In-DOTA-TATE. KEYWORDS: Protein binding, radiofarmaceuticals, equilibrium dialysis, 90Y-DOTA-TATE, 111In- DOTA-TATE

  12. Analysis of the relationship between interleukin polymorphisms within miRNA-binding regions and alcoholic liver disease.

    Science.gov (United States)

    Novo-Veleiro, I; Cieza-Borrella, C; Pastor, I; González-Sarmiento, R; Laso, F-J; Marcos, M

    2018-05-01

    Alcohol consumption promotes inflammation through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-?B pathway, leading to organic damage. Some micro-RNA (miRNA) molecules modulate this inflammatory response by downregulating TLR4/NF-?B pathway mediators, like interleukins (ILs). Thus, polymorphisms within IL genes located near miRNA binding sites could modify the risk of ethanol-induced damage. The present study analyzed potential relationships between alcoholism or alcoholic liver disease (ALD) and IL12B 2124 G>T (rs1368439), IL16 5000 C>T (rs1131445), IL1R1 3114 C>T (rs3917328), and NFKB1 3400 A>G (rs4648143) polymorphisms. The study included 301 male alcoholic patients and 156 male healthy volunteers. Polymorphisms were genotyped using TaqMan ® PCR assays for allelic discrimination. Allele and genotype frequencies were compared between groups. Logistic regression analysis was performed to analyze the inheritance model. Analysis of the IL1R1 (rs3917328) polymorphism showed that the proportion of alleleT carriers (CT and TT genotypes) was higher in healthy controls (9.7%) than in alcoholic patients (6.5%; P=.042). However, multivariable logistic regression analyses did not yield a significant result. No differences between groups were found for other analyzed polymorphisms. Our study describes, for the first time, the expected frequencies of certain polymorphisms within miRNA-binding sites in alcoholic patients with and without ALD. Further studies should be developed to clarify the potential relevance of these polymorphisms in alcoholism and ALD development. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

  13. The impact of policies regulating alcohol trading hours and days on specific alcohol-related harms: a systematic review.

    Science.gov (United States)

    Sanchez-Ramirez, Diana C; Voaklander, Donald

    2018-02-01

    Evidence supports the expectation that changes in time of alcohol sales associate with changes in alcohol-related harm in both directions. However, to the best of our knowledge, no comprehensive systematic reviews had examined the effect of policies restricting time of alcohol trading on specific alcohol-related harms. To compile existing evidence related to the impact of policies regulating alcohol trading hours/days of on specific harm outcomes such as: assault/violence, motor vehicle crashes/fatalities, injury, visits to the emergency department/hospital, murder/homicides and crime. Systematic review of literature studying the impact of policies regulation alcohol trading times in alcohol-related harm, published between January 2000 and October 2016 in English language. Results support the premise that policies regulating times of alcohol trading and consumption can contribute to reduce injuries, alcohol-related hospitalisations/emergency department visits, homicides and crime. Although the impact of alcohol trading policies in assault/violence and motor vehicle crashes/fatalities is also positive, these associations seem to be more complex and require further study. Evidence suggests a potential direct effect of policies that regulate alcohol trading times in the prevention of injuries, alcohol-related hospitalisations, homicides and crime. The impact of these alcohol trading policies in assault/violence and motor vehicle crashes/fatalities is less compelling. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  14. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

    KAUST Repository

    Li, Lixin

    2013-07-01

    Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

  15. Specific binding of beta-endorphin to normal human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  16. Face processing in chronic alcoholism: a specific deficit for emotional features.

    Science.gov (United States)

    Maurage, P; Campanella, S; Philippot, P; Martin, S; de Timary, P

    2008-04-01

    It is well established that chronic alcoholism is associated with a deficit in the decoding of emotional facial expression (EFE). Nevertheless, it is still unclear whether this deficit is specifically for emotions or due to a more general impairment in visual or facial processing. This study was designed to clarify this issue using multiple control tasks and the subtraction method. Eighteen patients suffering from chronic alcoholism and 18 matched healthy control subjects were asked to perform several tasks evaluating (1) Basic visuo-spatial and facial identity processing; (2) Simple reaction times; (3) Complex facial features identification (namely age, emotion, gender, and race). Accuracy and reaction times were recorded. Alcoholic patients had a preserved performance for visuo-spatial and facial identity processing, but their performance was impaired for visuo-motor abilities and for the detection of complex facial aspects. More importantly, the subtraction method showed that alcoholism is associated with a specific EFE decoding deficit, still present when visuo-motor slowing down is controlled for. These results offer a post hoc confirmation of earlier data showing an EFE decoding deficit in alcoholism by strongly suggesting a specificity of this deficit for emotions. This may have implications for clinical situations, where emotional impairments are frequently observed among alcoholic subjects.

  17. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    International Nuclear Information System (INIS)

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-01-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

  18. Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

    Science.gov (United States)

    Song, Wei; Guo, Jun-Tao

    2015-01-01

    Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

  19. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    Science.gov (United States)

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  20. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  1. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    Science.gov (United States)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  2. Alcohol exposure decreases CREB binding protein expression and histone acetylation in the developing cerebellum.

    Directory of Open Access Journals (Sweden)

    Weixiang Guo

    Full Text Available Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.We demonstrate that CREB binding protein (CBP is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

  3. Quantification of specific bindings of biomolecules by magnetorelaxometry

    Directory of Open Access Journals (Sweden)

    Steinhoff Uwe

    2008-03-01

    Full Text Available Abstract The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP, to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX. Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

  4. [3H]-ouabain binding to peripheral organs of cats: effect of ethanol

    International Nuclear Information System (INIS)

    Banerjee, S.P.; Sharma, V.K.

    1978-01-01

    The specific [ 3 H]-ouabain binding to microsomal fractions derived from cat heart, liver, spleen, and kidney increased significantly following chronic administration of ethanol. Since ouabain binds exclusively to cell membrane (Na + + K + )-adenosine triphosphatase ((Na + + K + )-ATPase), these results provide evidence for an increase in number of (Na + + K + )-ATPase macromolecules during chronic alcoholism. The importance of the increase in number of (Na + + K + )-ATPase molecules in the adaptive increase in ethanol metabolism and cardiac myopathy in chronic alcoholism is discussed. (author)

  5. Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

    Science.gov (United States)

    Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

    2009-09-01

    A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

  6. B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

    International Nuclear Information System (INIS)

    Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

    1990-01-01

    B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

  7. Preliminary studies of 99mTc-PQQ-NMDAR binding and effect of specificity binding by mannitol

    International Nuclear Information System (INIS)

    Xingqin Zhou; Yanyan Kong; Guoxian Cao; Jiankang Zhang

    2013-01-01

    Pyrroloquinoline quinone (PQQ) is a powerful neuroprotectant that specifically binds to brain NMDA receptors and inhibits excitotoxicity. Imaging this binding reaction in the brain remains a long sought goal in this field of study, and one of the primary challenges remaining is enabling soluble labeled PQQ to pass the blood-brain barrier (BBB). Previously, our group successfully labeled PQQ with Technetium-99m ( 99m Tc), a metastable nuclear isomer used in radioactive isotope medical tests. In this work, we determined the specific binding of 99m Tc-PQQ and NMDAR by radioligand receptor assay. Ebselen (EB) and MK-801 both effectively inhibited 99m Tc-PQQ binding. We then investigated methods of opening the BBB using mannitol to enable entry to the brain by 99m Tc-PQQ. Our results showed that 7.5 mL/kg of 20 % mannitol effectively opened the BBB and 20 min was the optimum treatment time. Competition studies showed that mannitol did not affect the specific binding between 99m Tc-PQQ and NMDA receptors. Using this method, the amount of 99m Tc-PQQ uptake and retention was increased most significantly in the hippocampus and cortex, and re-opening the BBB did not affect binding. Together, our results demonstrate that the use of mannitol to open the BBB may contribute significantly to improving image quality by increasing the uptake amount of a water-soluble agent in brain. (author)

  8. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  9. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    NARCIS (Netherlands)

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of

  10. Saturation of retinol-binding protein correlates closely to the severity of alcohol-induced liver disease

    DEFF Research Database (Denmark)

    Wagnerberger, S.; Schäfer, C.; Bode, C.

    2006-01-01

    Impaired metabolism of retinol has been shown to occur in alcohol-induced liver disease (ALD). The purpose of the present study was to investigate the saturation of retinol-binding protein (RBP) in 6 patients with different stages of ALD. Hospitalized alcohol consumers (n=118) with different stages...... chromatography and enzyme-linked immunosorbent assay methods, respectively. No differences were noted in daily retinol intake, but subjects with ALD had significantly lower concentrations of retinol in plasma (ALD1: 1.81+/-0.17 micromol/l [mean+/-S.E.M.]; ALD2: 1.95+/-0.24 micromol/l; ALD3: 0.67+/-0.13 micromol......: 43.5+/-6.2%; ALD3: 29.0+/-5.1%). The present study indicates that plasma concentrations of retinol and RBP per se do not correlate to severity of ALD, but rather that the retinol/RBP ratio links to the severity of alcohol-induced liver damage. From these results, a reduced availability of retinol...

  11. Effects of alcohol feeding on androgen receptors in the rat pituitary gland

    International Nuclear Information System (INIS)

    Chung, K.W.

    1987-01-01

    Specific binding of testosterone-1β,2β- 3 H by cytosol from anterior pituitary gland of ethanol-fed, isocaloric control, and castrated control and ethanol-fed rats with or without testosterone treatment has been investigated by charcoal assay. The number of androgen binding sites was significantly reduced in alcohol-fed rats when compared to the isocaloric control value, with no significant change in Kd. Castration significantly increased the number of receptor sites in control rats and when castrated control animals were treated with testosterone the binding sites were decreased to the intact control level. In contrast, castration or testosterone given to castrated alcohol-fed rats did not alter alcohol-induced reduction of the receptor sites. The binding affinity (Kd) is identical in all groups. The concentration of serum luteinizing hormone (LH) was significantly lower in alcohol-fed rats when compared to that of normal controls. An increased serum LH level with a decreased testosterone level was noted in castrated control rats. However, castration of alcohol-fed rats had little or no effects on the concentrations of LH and testosterone. Administration of testosterone suppressed castration-induced high LH in control rats but alcohol induced reduction of LH level was not altered by this treatment. These findings indicate that alcohol exerts a suppressive effect on the content of androgen receptors and secretory functions of gonadotropins in the pituitary gland. 23 references, 1 figure, 1 table

  12. Decreased prefrontal cortical dopamine transmission in alcoholism.

    Science.gov (United States)

    Narendran, Rajesh; Mason, Neale Scott; Paris, Jennifer; Himes, Michael L; Douaihy, Antoine B; Frankle, W Gordon

    2014-08-01

    Basic studies have demonstrated that optimal levels of prefrontal cortical dopamine are critical to various executive functions such as working memory, attention, inhibitory control, and risk/reward decisions, all of which are impaired in addictive disorders such as alcoholism. Based on this and imaging studies of alcoholism that have demonstrated less dopamine in the striatum, the authors hypothesized decreased dopamine transmission in the prefrontal cortex in persons with alcohol dependence. To test this hypothesis, amphetamine and [11C]FLB 457 positron emission tomography were used to measure cortical dopamine transmission in 21 recently abstinent persons with alcohol dependence and 21 matched healthy comparison subjects. [11C]FLB 457 binding potential, specific compared to nondisplaceable uptake (BPND), was measured in subjects with kinetic analysis using the arterial input function both before and after 0.5 mg kg-1 of d-amphetamine. Amphetamine-induced displacement of [11C]FLB 457 binding potential (ΔBPND) was significantly smaller in the cortical regions in the alcohol-dependent group compared with the healthy comparison group. Cortical regions that demonstrated lower dopamine transmission in the alcohol-dependent group included the dorsolateral prefrontal cortex, medial prefrontal cortex, orbital frontal cortex, temporal cortex, and medial temporal lobe. The results of this study, for the first time, unambiguously demonstrate decreased dopamine transmission in the cortex in alcoholism. Further research is necessary to understand the clinical relevance of decreased cortical dopamine as to whether it is related to impaired executive function, relapse, and outcome in alcoholism.

  13. Substrate specificity of flavin-dependent vanillyl-alcohol oxidase from Penicillium simplicissimum.Evidence for the production of 4-hydroxycinnamyl alcohols from 4-allylphenols

    OpenAIRE

    Fraaije, Marco W.; Veeger, Cees; Berkel, Willem J.H. van

    1995-01-01

    The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyph...

  14. Effects of alcohol consumption on the allergen-specific immune response in mice

    DEFF Research Database (Denmark)

    Linneberg, Allan; Roursgaard, Martin; Hersoug, Lars-Georg

    2008-01-01

    There is evidence that chronic alcohol consumption impairs the T-helper 1 (Th1) lymphocyte-regulated cell-mediated immune response possibly favoring a Th2 deviation of the immune response. Moreover, a few epidemiological studies have linked alcohol consumption to allergen-specific IgE sensitization....

  15. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  16. The selectively bred high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats differ in sensitivity to nicotine.

    Science.gov (United States)

    de Fiebre, NancyEllen C; Dawson, Ralph; de Fiebre, Christopher M

    2002-06-01

    Studies in rodents selectively bred to differ in alcohol sensitivity have suggested that nicotine and ethanol sensitivities may cosegregate during selective breeding. This suggests that ethanol and nicotine sensitivities may in part be genetically correlated. Male and female high alcohol sensitivity (HAS), control alcohol sensitivity, and low alcohol sensitivity (LAS) rats were tested for nicotine-induced alterations in locomotor activity, body temperature, and seizure activity. Plasma and brain levels of nicotine and its primary metabolite, cotinine, were measured in these animals, as was the binding of [3H]cytisine, [3H]epibatidine, and [125I]alpha-bungarotoxin in eight brain regions. Both replicate HAS lines were more sensitive to nicotine-induced locomotor activity depression than the replicate LAS lines. No consistent HAS/LAS differences were seen on other measures of nicotine sensitivity; however, females were more susceptible to nicotine-induced seizures than males. No HAS/LAS differences in nicotine or cotinine levels were seen, nor were differences seen in the binding of nicotinic ligands. Females had higher levels of plasma cotinine and brain nicotine than males but had lower brain cotinine levels than males. Sensitivity to a specific action of nicotine cosegregates during selective breeding for differential sensitivity to a specific action of ethanol. The differential sensitivity of the HAS/LAS rats is due to differences in central nervous system sensitivity and not to pharmacokinetic differences. The differential central nervous system sensitivity cannot be explained by differences in the numbers of nicotinic receptors labeled in ligand-binding experiments. The apparent genetic correlation between ethanol and nicotine sensitivities suggests that common genes modulate, in part, the actions of both ethanol and nicotine and may explain the frequent coabuse of these agents.

  17. Russia-specific relative risks and their effects on the estimated alcohol-attributable burden of disease.

    Science.gov (United States)

    Shield, Kevin D; Rehm, Jürgen

    2015-05-10

    Alcohol consumption is a major risk factor for the burden of disease globally. This burden is estimated using Relative Risk (RR) functions for alcohol from meta-analyses that use data from all countries; however, for Russia and surrounding countries, country-specific risk data may need to be used. The objective of this paper is to compare the estimated burden of alcohol consumption calculated using Russia-specific alcohol RRs with the estimated burden of alcohol consumption calculated using alcohol RRs from meta-analyses. Data for 2012 on drinking indicators were calculated based on the Global Information System on Alcohol and Health. Data for 2012 on mortality, Years of Life Lost, Years Lived with Disability, and Disability-Adjusted Life Years (DALYs) lost by cause were obtained by country from the World Health Organization. Alcohol Population-Attributable Fractions (PAFs) were calculated based on a risk modelling methodology from Russia. These PAFs were compared to PAFs calculated using methods applied for all other countries. The 95% Uncertainty Intervals (UIs) for the alcohol PAFs were calculated using a Monte Carlo-like method. Using Russia-specific alcohol RR functions, in Russia in 2012 alcohol caused an estimated 231,900 deaths (95% UI: 185,600 to 278,200) (70,800 deaths among women and 161,100 deaths among men) and 13,295,000 DALYs lost (95% UI: 11,242,000 to 15,348,000) (3,670,000 DALYs lost among women and 9,625,000 DALYs lost among men) among people 0 to 64 years of age. This compares to an estimated 165,600 deaths (95% UI: 97,200 to 228,100) (29,700 deaths among women and 135,900 deaths among men) and 10,623,000 DALYs lost (95% UI: 7,265,000 to 13,754,000) (1,783,000 DALYs lost among women and 8,840,000 DALYs lost among men) among people 0 to 64 years of age caused by alcohol when non-Russia-specific alcohol RRs were used. Results indicate that if the Russia-specific RRs are used when estimating the health burden attributable to alcohol consumption in

  18. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  19. Are Alcohol Taxation and Pricing Policies Regressive? Product-Level Effects of a Specific Tax and a Minimum Unit Price for Alcohol.

    Science.gov (United States)

    Vandenberg, Brian; Sharma, Anurag

    2016-07-01

    To compare estimated effects of two policy alternatives, (i) a minimum unit price (MUP) for alcohol and (ii) specific (per-unit) taxation, upon current product prices, per capita spending (A$), and per capita consumption by income quintile, consumption quintile and product type. Estimation of baseline spending and consumption, and modelling policy-to-price and price-to-consumption effects of policy changes using scanner data from a panel of demographically representative Australian households that includes product-level details of their off-trade alcohol spending (n = 885; total observations = 12,505). Robustness checks include alternative price elasticities, tax rates, minimum price thresholds and tax pass-through rates. Current alcohol taxes and alternative taxation and pricing policies are not highly regressive. Any regressive effects are small and concentrated among heavy consumers. The lowest-income consumers currently spend a larger proportion of income (2.3%) on alcohol taxes than the highest-income consumers (0.3%), but the mean amount is small in magnitude [A$5.50 per week (95%CI: 5.18-5.88)]. Both a MUP and specific taxation will have some regressive effects, but the effects are limited, as they are greatest for the heaviest consumers, irrespective of income. Among the policy alternatives, a MUP is more effective in reducing consumption than specific taxation, especially for consumers in the lowest-income quintile: an estimated mean per capita reduction of 11.9 standard drinks per week (95%CI: 11.3-12.6). Policies that increase the cost of the cheapest alcohol can be effective in reducing alcohol consumption, without having highly regressive effects. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  20. The influence of surface integrin binding patterns on specific biomaterial-cell interactions

    Science.gov (United States)

    Beranek, Maggi Marie

    As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

  1. [HPLC Specific Fingerprint of Alcohol Extract of Andrographis paniculata].

    Science.gov (United States)

    Liu, Fei-fei; Fan, Chun-lin; Huang, Xiao-jun; Wang, Gui-yang; Wang, Ying; Ye, Wen-cai

    2015-07-01

    To establish the specific fingerprint of alcohol extract of Andrographis paniculata by HPLC. The analysis was performed on Cosmosil 5C18 -MS-II (250 mm x 4. 6 mm, 5 µm) column, with gradient phase consisting of acetonitrile and water at the flow rate of 1. 0 mL/min. The UV detection wavelength was set at 225 nm, and column temperature was 30 °C. The specific fingerprint chromatogram was established and seven common peaks were identified by comparison with the reference standards and LC-MS. The relative retention times were 1. 00 (No. 1, andrographolide), 1. 04 (No. 2, deoxyandrographoside), 1. 07 (No. 3, isoandrographolide), 1. 10 (No. 4, 14-deoxy-11, 12-didehydroandrographoside), 1. 50 (No. 5, neoandrographolide), 1. 75 ( No. 6, deoxyandrographolide) and 1. 79 (No. 7, dehydroandrographolide). The method is simple and reproducible with high precision, which can provide the basis for the quality control and evaluation of alcohol extract of Andrographis paniculata and its preparations.

  2. DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

    Science.gov (United States)

    Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

    1995-09-05

    Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

  3. LUSH-based SPR sensor for the detection of alcohols and pheromone

    Science.gov (United States)

    Lau, Hui-Chong; Lee, Yeon-Kyung; Kwon, Jae-Young; Sohn, Young-Soo; Lim, Jeong Ok

    2013-05-01

    Protein is a widely used sensing substrate in the biosensing technology. In the study conducted here, we used odorant binding protein, LUSH from Drosophila as a biosensing substrate in a miniaturized surface plasmon resonance (SPR) sensor. LUSH contains the specific alcohols binding sites, which mediates the detection of alcohols and pheromone. We first modified the surface of the gold sensor chip using the self assembled monolayer in the chloroform solution. The saturated concentration was determined prior to the detection of alcohols and pheromone at various concentrations. The results showed that the LUSH was saturated at 1000 μg/ml on the gold sensor chip. The detection response of LUSH was significant at higher concentration of alcohols. LUSH detected ethanol at concentration >=50% propanol was detected at >=25% whereas pheromone was detected at >=1.25 μg/μl. The results provide some fundamental information on the potential use of LUSH-based SPR as a simple and easy protein-based sensor in the near future.

  4. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  5. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    Science.gov (United States)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  6. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  7. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  8. Daily affect variability and context-specific alcohol consumption.

    Science.gov (United States)

    Mohr, Cynthia D; Arpin, Sarah; McCabe, Cameron T

    2015-11-01

    Research explored the effects of variability in negative and positive affect on alcohol consumption, specifying daily fluctuation in affect as a critical form of emotion dysregulation. Using daily process methodology allows for a more objective calculation of affect variability relative to traditional self-reports. The present study models within-person negative and positive affect variabilities as predictors of context-specific consumption (i.e. solitary vs. social drinking), controlling for mean levels of affect. A community sample of moderate-to-heavy drinkers (n = 47; 49% women) from a US metropolitan area reported on affect and alcohol consumption thrice daily for 30 days via a handheld electronic interviewer. Within-person affect variability was calculated using daily standard deviations in positive and negative affect. Within person, greater negative and positive variabilities are related to greater daily solitary and social consumption. Across study days, mean levels of negative and positive affect variabilities related to greater social consumption between persons; yet, aggregated negative affect variability was related to less solitary consumption. Results affirm affect variability as a unique predictor of alcohol consumption, independent of mean affect levels. Yet, it is important to differentiate social context of consumption, as well as type of affect variability, particularly at the between-person level. These distinctions help clarify inconsistencies in the self-medication literature regarding associations between average levels of affect and consumption. Importantly, consistent within-person relationships for both variabilities support arguments that both negative and positive affect variabilities are detrimental and reflect an inability to regulate emotional experience. © 2015 Australasian Professional Society on Alcohol and other Drugs.

  9. Sport-related achievement motivation and alcohol outcomes: an athlete-specific risk factor among intercollegiate athletes.

    Science.gov (United States)

    Weaver, Cameron C; Martens, Matthew P; Cadigan, Jennifer M; Takamatsu, Stephanie K; Treloar, Hayley R; Pedersen, Eric R

    2013-12-01

    Intercollegiate athletes report greater alcohol consumption and more alcohol-related problems than their non-athlete peers. Although college athletes share many of the same problems faced by non-athletes, there are some consequences that are unique to athletes. Studies have demonstrated that alcohol negatively affects athletic performance including increased dehydration, impeded muscle recovery, and increased risk for injury. Beyond risk factors for alcohol misuse that may affect college students in general, research has begun to examine risk factors that are unique to collegiate athletes. For example, research has found that off-season status, the leadership role, and athlete-specific drinking motives are associated with increased alcohol use. Given these findings, it is possible that other athlete-specific variables influence alcohol misuse. One such variable may be sport achievement orientation. The purpose of the current study was to examine the relationship between sport achievement orientation and alcohol outcomes. Given previous research regarding seasonal status and gender, these variables were examined as moderators. Varsity athletes (n=263) completed the Sport Orientation Questionnaire, which assesses sport-related achievement orientation on three scales (Competitiveness, Win Orientation, and Goal Orientation). In addition, participants completed measures of alcohol use and alcohol-related problems. Results indicated that Competitiveness, Win Orientation, and Goal Orientation were all significantly associated with alcohol use, but not alcohol-related problems. Moreover, these relationships were moderated by seasonal status and gender. These interactions, clinical implications, and limitations are discussed. © 2013.

  10. Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study.

    Directory of Open Access Journals (Sweden)

    Carlos Navarro-Retamal

    Full Text Available Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT (EC 2.3.1.84 catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This

  11. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  12. Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

    Directory of Open Access Journals (Sweden)

    Salvatore Loguercio

    2018-03-01

    Full Text Available CCCTC-binding factor (CTCF is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells and developmental stage-specificity (pre-B vs. pro-B in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for

  13. Trait Anger and Partner-Specific Anger Management Moderate the Temporal Association Between Alcohol Use and Dating Violence.

    Science.gov (United States)

    Shorey, Ryan C; McNulty, James K; Moore, Todd M; Stuart, Gregory L

    2017-03-01

    Research demonstrates alcohol temporally precedes and increases the odds of violence between intimate partners. However, despite an extensive theoretical literature on factors that likely moderate the relationship between alcohol and dating violence, minimal empirical research has examined such moderators. The purpose of the present study was to examine two potential moderators of this association: trait anger and partner-specific anger management. Undergraduate men (N = 67) who had consumed alcohol within the past month and were in current dating relationships completed a baseline assessment of their trait anger and partner-specific anger management skills and subsequently completed daily assessments of their alcohol use and violence perpetration (psychological, physical, and sexual) for up to 90 consecutive days. Alcohol was significantly associated with increased odds of physical aggression among men with relatively high but not low trait anger and partner-specific anger management deficits. In contrast, alcohol was significantly associated with increased odds of sexual aggression among men with relatively low trait anger and partner-specific anger management deficits. Our findings demonstrate important differences in the roles of acute intoxication and anger management in the risk of physical aggression and sexual dating violence. Interventions for dating violence may benefit from targeting both alcohol and adaptive anger management skills.

  14. Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

    Science.gov (United States)

    Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

    2013-02-15

    PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

  15. Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

    International Nuclear Information System (INIS)

    Ferron, P.M.; Banner, W. Jr.; Duckles, S.P.

    1984-01-01

    In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha 1 adrenergic ligand, ( 3 H) prazosin, were studied in blood vessel homogenates. No specific ( 3 H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery 3 H-prazosin binding was saturable with a K/sub d/ of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, [ 3 H](-) quinuclidinyl benzilate (QNB) and one for alpha 2 adrenergic receptors, ( 3 H) yohimbine. The results of these studies demonstrate a lack of alpha 1 adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine. 29 references, 3 figures, 2 tables

  16. Alcohol-specific parenting, adolescents' self-control, and alcohol use : A moderated mediation model

    NARCIS (Netherlands)

    Koning, Ina M.; van den Eijnden, Regina J J M; Vollebergh, Wilma A M

    2014-01-01

    Objective: There is convincing evidence that parental rules about alcohol are important in curbing adolescents' alcohol use. However, little is known about the mechanisms through which the direct link between alcohol-specifi c parenting and alcohol use is obtained. In this study, we investigated the

  17. Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

    Directory of Open Access Journals (Sweden)

    Tiffiny Rye-McCurdy

    2016-09-01

    Full Text Available Retroviruses specifically package full-length, dimeric genomic RNA (gRNA even in the presence of a vast excess of cellular RNA. The “psi” (Ψ element within the 5′-untranslated region (5′UTR of gRNA is critical for packaging through interaction with the nucleocapsid (NC domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1 Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity.

  18. Anticipating and addressing event-specific alcohol consumption among adolescents.

    Science.gov (United States)

    Pettigrew, Simone; Biagioni, Nicole; Jongenelis, Michelle I

    2016-07-29

    Various specific events and celebrations are associated with excessive alcohol consumption and related harms. End-of-school celebrations such as Schoolies in Australia are of particular concern given high levels of documented harm among underage and young drinkers. The present study investigated high school students' expectations of their Schoolies celebrations to inform future interventions to reduce adverse outcomes among members of this vulnerable group and other young people involved in similar rites of passage. A link to an online survey was distributed via high schools and Schoolies-related websites. The survey included qualitative questions that invited respondents to discuss (i) aspects of Schoolies they were looking forward to most and least and (ii) their perceptions of the likely consequences if they refrained from consuming alcohol during the event. In total, 435 students provided responses. Respondents discussed the role of Schoolies in marking their transition to adulthood. Their comments revealed a cross-temporal focus indicating that Schoolies is simultaneously symbolic of the past, present, and future. Through its ability to enhance social interaction, alcohol was perceived to have a vital role in realising the potential of this event to signify and facilitate this temporal progression. Results suggest interventions that treat Schoolies as an isolated event that occurs in specific locations may fail to appreciate the extent to which these events transcend time for those involved. Instead, harm reduction is likely to involve a reconceptualisation of the event among both participants and authority figures to facilitate the provision of alternative pastimes to drinking during Schoolies that yield similar social benefits.

  19. Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition.

    Directory of Open Access Journals (Sweden)

    Matthew W Parker

    Full Text Available Neuropilin (Nrp receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF family of proangiogenic cytokines and the semaphorin 3 (Sema3 family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

  20. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Science.gov (United States)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  1. The PP1 binding code: a molecular-lego strategy that governs specificity.

    Science.gov (United States)

    Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

    2013-01-01

    Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

  2. Cross-modal working memory binding and word recognition skills: how specific is the link?

    Science.gov (United States)

    Wang, Shinmin; Allen, Richard J

    2018-04-01

    Recent research has suggested that the creation of temporary bound representations of information from different sources within working memory uniquely relates to word recognition abilities in school-age children. However, it is unclear to what extent this link is attributable specifically to the binding ability for cross-modal information. This study examined the performance of Grade 3 (8-9 years old) children on binding tasks requiring either temporary association formation of two visual items (i.e., within-modal binding) or pairs of visually presented abstract shapes and auditorily presented nonwords (i.e., cross-modal binding). Children's word recognition skills were related to performance on the cross-modal binding task but not on the within-modal binding task. Further regression models showed that cross-modal binding memory was a significant predictor of word recognition when memory for its constituent elements, general abilities, and crucially, within-modal binding memory were taken into account. These findings may suggest a specific link between the ability to bind information across modalities within working memory and word recognition skills.

  3. Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

    OpenAIRE

    Parsons, C A; West, S C

    1990-01-01

    T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

  4. Anticipating and addressing event-specific alcohol consumption among adolescents

    Directory of Open Access Journals (Sweden)

    Simone Pettigrew

    2016-07-01

    Full Text Available Abstract Background Various specific events and celebrations are associated with excessive alcohol consumption and related harms. End-of-school celebrations such as Schoolies in Australia are of particular concern given high levels of documented harm among underage and young drinkers. The present study investigated high school students’ expectations of their Schoolies celebrations to inform future interventions to reduce adverse outcomes among members of this vulnerable group and other young people involved in similar rites of passage. Methods A link to an online survey was distributed via high schools and Schoolies-related websites. The survey included qualitative questions that invited respondents to discuss (i aspects of Schoolies they were looking forward to most and least and (ii their perceptions of the likely consequences if they refrained from consuming alcohol during the event. In total, 435 students provided responses. Results Respondents discussed the role of Schoolies in marking their transition to adulthood. Their comments revealed a cross-temporal focus indicating that Schoolies is simultaneously symbolic of the past, present, and future. Through its ability to enhance social interaction, alcohol was perceived to have a vital role in realising the potential of this event to signify and facilitate this temporal progression. Conclusions Results suggest interventions that treat Schoolies as an isolated event that occurs in specific locations may fail to appreciate the extent to which these events transcend time for those involved. Instead, harm reduction is likely to involve a reconceptualisation of the event among both participants and authority figures to facilitate the provision of alternative pastimes to drinking during Schoolies that yield similar social benefits.

  5. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  6. Specificity of cellular DNA-binding sites of microbial populations in a Florida reservoir

    International Nuclear Information System (INIS)

    Paul, J.H.; Pichard, S.L.

    1989-01-01

    The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [ 3 H]- or [ 32 P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nuclei acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments

  7. RNA binding specificity of Ebola virus transcription factor VP30.

    Science.gov (United States)

    Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

    2016-09-01

    The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

  8. Correlation between (3H)dopamine specific uptake and (3H)GBR 12783 specific binding during the maturation of rat striatum

    International Nuclear Information System (INIS)

    Bonnet, J.J.; Costentin, J.

    1989-01-01

    The development of the specific uptake of dopamine in the rat striatum during the early postnatal period is compared with the ontogenetic changes of the specific binding of ( 3 H)GBR 12783 to the site of uptake inhibition. During maturation, the increase in the specific binding of ( 3 H)GBR 12783 parallels the increase in the specific uptake of dopamine. ( 3 H)GBR 12783 specific binding sites increase in number from day 1 postpartum until 40 days, when they reach the adult level. In 40 day-old rats, the weight of the striatum represents 80% of adult values. The affinity of ( 3 H)GBR 12783 for the inhibition site is similar in membrane preparations obtained from 6 day-old pups and adults; this results in a same ability of the inhibitor to block the specific uptake of dopamine into synaptosomes obtained from pups or adult rats. These data support the hypothesis of the existence of a single molecular entity including both the inhibition site and the carrier itself

  9. Structural Determinants of Specific Lipid Binding to Potassium Channels

    NARCIS (Netherlands)

    Weingarth, M.H.|info:eu-repo/dai/nl/330985655; Prokofyev, A.; van der Cruijsen, E.A.W.|info:eu-repo/dai/nl/330826743; Nand, D.|info:eu-repo/dai/nl/337731403; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Pongs, O.; Baldus, M.|info:eu-repo/dai/nl/314410864

    2013-01-01

    We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsAKv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while

  10. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  11. Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

    Science.gov (United States)

    Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

    2018-03-27

    Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    International Nuclear Information System (INIS)

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

    1990-01-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

  13. Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

    Science.gov (United States)

    van Andel, Esther; de Bus, Ian; Tijhaar, Edwin J; Smulders, Maarten M J; Savelkoul, Huub F J; Zuilhof, Han

    2017-11-08

    Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

  14. Beverage-Specific Alcohol Sale and Cardiovascular Mortality in Russia

    Directory of Open Access Journals (Sweden)

    Y. E. Razvodovsky

    2010-01-01

    Full Text Available Objective. Recent research evidence suggests that the consumption of different types of alcoholic beverage may have a differential effect on cardiovascular diseases (CVD mortality rates. The aim of this study was to examine the relation between the consumption of different beverage types and CVD mortality rates in Russia across the later-Soviet and post-Soviet periods. Method. Age-standardized male and female CVD mortality data for the period 1970–2005 and data on beverage-specific alcohol sales were obtained Russian State Statistical Committee (Rosstat. Time-series analytical modeling techniques (ARIMA were used to examine the relation between the sales of different alcoholic beverages and CVD mortality rates. Results. Vodka consumption as measured by sales was significantly associated with both male and female CVD mortality rates: a 1 liter increase in vodka sales would result in a 5.3% increase in the male CVD mortality rate and a 3.7% increase in the female rate. The consumption of beer and wine were not associated with CVD mortality rates. Conclusions. The findings from this study suggest that public health efforts should focus on both reducing overall consumption and changing beverage preference away from distilled spirits in order to reduce cardiovascular mortality rates in Russia.

  15. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  16. Presence of specific growth hormone binding sites in rainbow trout (Oncorhynchus mykiss) tissues: characterization of the hepatic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yao, K.; Niu, P.D.; Le Gac, F.; Le Bail, P.Y. (Laboratoire de Physiologie des Poissons, INRA, Rennes, (France))

    1991-01-01

    The present work outlines the presence of specific binding for chinook salmon growth hormone (sGH) in different tissue preparations of rainbow trout. Optimal incubation conditions (pH, Tris, MgCl{sub 2}) were determined. Specific binding was very sensitive to salt concentration during incubation. The specific binding reached a plateau after 15 and 25 hr of incubation at 12 and 4 {degree}. At 20 {degree}, specific and nonspecific binding were not stable. Specific binding dissociation was slower than association and was only partial. The binding was saturable (Bmax = 187 +/- 167 pmol), of high affinity (Ka = 2.4 +/- 0.8 10(9) M-1), and very specific for GH, properties which are in agreement with the characteristics of hormonal receptors. Sea bream and mammalian GH appeared 2- and 30-fold, respectively, less potent than cold sGH2 for displacing {sup 125}I-sGH2. Tissue preparations from ovary, testis, fat, skin, cartilage, gill, blood pellet, brain, spleen, kidney, and muscle showed significant saturable binding.

  17. Presence of specific growth hormone binding sites in rainbow trout (Oncorhynchus mykiss) tissues: characterization of the hepatic receptor

    International Nuclear Information System (INIS)

    Yao, K.; Niu, P.D.; Le Gac, F.; Le Bail, P.Y.

    1991-01-01

    The present work outlines the presence of specific binding for chinook salmon growth hormone (sGH) in different tissue preparations of rainbow trout. Optimal incubation conditions (pH, Tris, MgCl 2 ) were determined. Specific binding was very sensitive to salt concentration during incubation. The specific binding reached a plateau after 15 and 25 hr of incubation at 12 and 4 degree. At 20 degree, specific and nonspecific binding were not stable. Specific binding dissociation was slower than association and was only partial. The binding was saturable (Bmax = 187 +/- 167 pmol), of high affinity (Ka = 2.4 +/- 0.8 10(9) M-1), and very specific for GH, properties which are in agreement with the characteristics of hormonal receptors. Sea bream and mammalian GH appeared 2- and 30-fold, respectively, less potent than cold sGH2 for displacing 125 I-sGH2. Tissue preparations from ovary, testis, fat, skin, cartilage, gill, blood pellet, brain, spleen, kidney, and muscle showed significant saturable binding

  18. The relationship between exposure to brand-specific alcohol advertising and brand-specific consumption among underage drinkers--United States, 2011-2012.

    Science.gov (United States)

    Siegel, Michael; Ross, Craig S; Albers, Alison B; DeJong, William; King, Charles; Naimi, Timothy S; Jernigan, David H

    2016-01-01

    Marketing is increasingly recognized as a potentially important contributor to youth drinking, yet few studies have examined the relationship between advertising exposure and alcohol consumption among underage youth at the brand level. To examine the relationship between brand-specific exposure to alcohol advertising among underage youth and the consumption prevalence of each brand in a national sample of underage drinkers. We analyzed the relationship between population-level exposure of underage youth ages 12-20 to brand-specific alcohol advertising in national magazines and television programs and the 30-day consumption prevalence--by brand--among a national sample of underage drinkers ages 13-20. Underage youth exposure to alcohol advertising by brand for each month in 2011, measured in gross rating points (GRPs, a standard measure of advertising exposure), was obtained from GfK MRI (a media consumer research company) and Nielsen for all measured national issues of magazines and all national television programs, respectively. The 30-day consumption prevalence for each brand was obtained from a national survey of 1031 underage drinkers conducted between December 2011 and May 2012. Underage youth were more than five times more likely to consume brands that advertise on national television and 36% more likely to consume brands that advertise in national magazines. The consumption prevalence of a brand increased by 36% for each 1.5 standard deviation (50 GRPs) increase in television adstock among underage youth and by 23% for each 1.5 standard deviation (10 GRPs) increase in magazine adstock. These findings suggest that alcohol advertising influences an important aspect of drinking behavior--brand choice--among youth who consume alcohol.

  19. The Relationship between Exposure to Brand-Specific Alcohol Advertising and Brand-Specific Consumption among Underage Drinkers—United States, 2011-2012

    Science.gov (United States)

    Siegel, Michael; Ross, Craig S.; Albers, Alison B.; DeJong, William; King, Charles; Naimi, Timothy S.; Jernigan, David H.

    2015-01-01

    Background Marketing is increasingly recognized as a potentially important contributor to youth drinking, yet few studies have examined the relationship between advertising exposure and alcohol consumption among underage youth at the brand level. Objectives To examine the relationship between brand-specific exposure to alcohol advertising among underage youth and the consumption prevalence of each brand in a national sample of underage drinkers. Methods We analyzed the relationship between population-level exposure of underage youth ages 12-20 to brand-specific alcohol advertising in national magazines and television programs and the 30-day consumption prevalence—by brand—among a national sample of underage drinkers ages 13-20. Underage youth exposure to alcohol advertising by brand for each month in 2011, measured in gross rating points (GRPs), was obtained from GfK MRI and Nielsen for all measured national issues of magazines and all national television programs, respectively. The 30-day consumption prevalence for each brand was obtained from a national survey of 1,031 underage drinkers conducted between December 2011 and May 2012. Results Underage youth were more than five times more likely to consume brands that advertise on national television and 36% more likely to consume brands that advertise in national magazines. The consumption prevalence of a brand increased by 36% for each 1.5 standard deviation (50 GRPs) increase in television adstock among underage youth and by 23% for each 1.5 standard deviation (10 GRPs) increase in magazine adstock. Conclusion These findings suggest that alcohol advertising influences an important aspect of drinking behavior— brand choice—among youth who consume alcohol. PMID:26479468

  20. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  1. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    Science.gov (United States)

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  2. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    International Nuclear Information System (INIS)

    Yin Huquan; Kim, Youn-Chul; Chung, Young-Suk; Kim, Young-Chul; Shin, Young-Kee; Lee, Byung-Hoon

    2009-01-01

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-α levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-α, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c

  3. The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

    Directory of Open Access Journals (Sweden)

    Anitha Shanmugham

    Full Text Available Redox enzyme maturation proteins (REMPs bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA and TorD (specific for trimethylamine N-oxide reductase, TorA. Green fluorescent protein (GFP was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

  4. Concentrations of lipopolysaccharide-binding protein, bactericidal/permeability-increasing protein, soluble CD14 and plasma lipids in relation to endotoxaemia in patients with alcoholic liver disease

    DEFF Research Database (Denmark)

    Schäfer, C.; Parlesak, Alexandr; Schütt, C.

    2002-01-01

    of endotoxin on its target cells (LPS-binding protein and sCD14) were increased. Endotoxin antagonists, such as bactericidal/permeability-increasing protein and high-density lipoprotein, were increased in the pre-cirrhotic stages, whereas a significant reduction of the latter was observed in cirrhosis. Low......-density lipoprotein remained unchanged. The elevation of binding factors in the pre-cirrhotic stages of alcoholic liver disease might attenuate the effects of endotoxaemia, whereas in cirrhosis the reduction of high density lipoprotein, to which large quantities of endotoxin bind, may contribute to its pro...

  5. 98 Specific IGE and IGG Binding to Allergoids of Phleum pratense

    Science.gov (United States)

    Cases, Barbara; Fernandez-Caldas, Enrique; Tudela, Jose Ignacio; Fernandez, Eva Abel; Sanchez-Garcia, Silvia; Ibañez, M. Dolores; Escudero, Carmelo; Casanovas, Miguel

    2012-01-01

    Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods The sera of 69 patients sensitized to P. pratense were tested. All these patients had signs and symptoms of rhinoconjunctivitis with, or without, asthma in May and June of 2011. All these patients had positive skin prick tests to a standardized extract of P. pratense, and other grass species. Most patients were also sensitized to olive pollen. Specific IgE and IgG binding were analysed by direct ELISA against P. pratense native (non-modified) and allergoid extracts. Relative potencies were evaluated through ELISA inhibition assays, and the protein composition of non-modified and allergoid samples was determined by Mass Spectrometry (MS/MS). Results Mean Specific IgE levels against the native extract was 16.68 ± 11.65 Units (U) and against the allergoid: 7.26 ± 8.24 U (P allergoid (P = 0.16; Mann-Whitney). Linear regression coefficients obtained between immunoglobulin reactivity against both extracts were: r2 = 0.51 for specific IgE and r2 = 0.83 for specific IgG. An important decrease in the allergenic activity, measured by inhibition ELISA, was clearly observed. The MS/MS assay revealed the presence of the mayor allergen, and some isoforms, in non-modified and allergoid extracts. Conclusions Results obtained demonstrate that the glutaraldehyde polymerization process induces an important decrease in specific IgE binding to allergoids of P. pratense while there are no significant differences in specific IgG binding. The allergenic composition of the P. pratense allergoid was

  6. The specific binding of the thyroid hormones to matrix isolated from rat liver nuclei

    International Nuclear Information System (INIS)

    Wilson, B.D.; Albrecht, C.F.; Wium, C.A.

    1982-01-01

    Specific binding sites for the thyroid hormones have been demonstrated in the liver nuclear matrix, a structural framework of the nucleus. When labelled 3,5,3'-tri-iodo-L-thyronine ([ 125 l]T 3 ) is injected into rats, 5% of the total nucleus bound T 3 is bound to the matrix after 1 hour. However, when either isolated nuclei or isolated nuclear matrices were incubated with[ 125 l]T 3 in vitro, a 3- to 7- fold greater number of specific T 3 binding sites were revealed in the nuclear matrix. The properties of the matrix-associated thyroid hormone binding sites were investigated in vitro. These binding sites showed limited capacity and high affinity for T 3 ; the equilibrium association constant (K(a)) was 1,3X10 M -1 and the binding capacity was 20,2 fmol T 3 per 100 μg matrix protein

  7. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    Science.gov (United States)

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  8. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  9. Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette

    2014-01-01

    MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide...... library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices...... representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide...

  10. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

    International Nuclear Information System (INIS)

    Mong, S.; Wu, H.L.; Clark, M.A.; Stadel, J.M.; Gleason, J.G.; Crooke, S.T.

    1984-01-01

    A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [ 3 H]-leukotriene D4 [( 3 H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for [ 3 H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [ 3 H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [ 3 H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [ 3 H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [ 3 H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung

  11. Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

    Science.gov (United States)

    Tsuda, M; Kurokawa, T; Takeuchi, M; Sugino, Y

    1975-10-01

    Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

  12. Detection of site-specific binding and co-binding of ligands to macromolecules using 19F NMR

    International Nuclear Information System (INIS)

    Jenkins, B.G.

    1991-01-01

    Study of ligand-macromolecular interactions by 19 F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of 19 F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple 19 F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of 19 F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed

  13. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

    2011-01-01

    a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

  14. The effect of chronic ethanol on glutamate binding in human and rat brain

    International Nuclear Information System (INIS)

    Cummins, J.T.; Sack, M.; von Hungen, K.

    1990-01-01

    Quantitative autoradiographic techniques demonstrate that chronic alcohol administration causes a decrease in [ 3 H]-glutamate binding to hippocampal N-methyl-D-aspartate (NMDA) receptors. A 14% decrease in [ 3 H]-glutamate binding in the hippocampal CA 1 region is seen both in the rat after five days of ethanol administration and in postmortem hippocampal tissues from alcoholics. In the rat, 24 hr ethanol withdrawal values are intermediate between control and alcohol binding levels. There was no significant effect of ethanol on [ 3 H]-glutamate binding in the cortex or caudate

  15. Substrate specificity and subcellular localization of the aldehyde-alcohol redox-coupling reaction in carp cones.

    Science.gov (United States)

    Sato, Shinya; Fukagawa, Takashi; Tachibanaki, Shuji; Yamano, Yumiko; Wada, Akimori; Kawamura, Satoru

    2013-12-20

    Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.

  16. Theoretical investigation of the weak interaction between graphene and alcohol solvents

    Science.gov (United States)

    Wang, Haining; Chen, Sian; Lu, Shanfu; Xiang, Yan

    2017-05-01

    The dispersion of graphene in five different alcohol solvents was investigated by evaluating the binding energy between graphene and alcohol molecules using DFT-D method. The calculation showed the most stable binding energy appeared at the distance of ∼3.5 Å between graphene and alcohol molecules and increased linearly as changing the alcohol from methanol to 1-pentanol. The weak interaction was further graphically illustrated using the reduced density gradient method. The theoretical study revealed alcohols with more carbon atoms could be a good starting point for screening suitable solvents for graphene dispersion.

  17. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M. (Univ. of Alabama at Birmingham (USA))

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37{degree}C. A functional fibrinogen-binding component (M{sub r}, 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with {sup 125}I-fibrinogen. Fibrinogen degradation did not occur at 4{degree}C but did occur at 22 and 37{degree}C. When bacteria and iodinated fibrinogen were incubated at 37{degree}C, two major fibrinogen fragments (M{sub r}, 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M{sub r}, 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M{sub r}-120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-{alpha}-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

  18. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    International Nuclear Information System (INIS)

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M.

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degree C. A functional fibrinogen-binding component (M r , 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125 I-fibrinogen. Fibrinogen degradation did not occur at 4 degree C but did occur at 22 and 37 degree C. When bacteria and iodinated fibrinogen were incubated at 37 degree C, two major fibrinogen fragments (M r , 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M r , 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M r -120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-α-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate

  19. Long-term reproducibility of in vivo measures of specific binding of radioligands in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Kilbourn, Michael R. E-mail: mkilbour@umich.edu

    2004-07-01

    The long-term reproducibility of measures of in vivo specific binding of radiolabeled forms of (+)-{alpha}-dihydrotetrabenazine (DTBZ) and d-threo-methylphenidate (MPH) in rat brain was examined. All studies were done using a consistent bolus plus infusion protocol and calculation of equilibrium distribution volume ratios (DVR). Over a period of eight years striatal DVR values for DTBZ binding to the vesicular monoamine transporter 2 (VMAT2) in young adult (8-10 wks old) rats showed very good reproducibility (3.62{+-}0.33, N=35). Equivalent values were obtained using either tritiated or carbon-11 labeled DTBZ, and were irrespective of sex of animals. Older animals (78 wks old) showed losses (-45%) of specific binding. Striatal binding of MPH to the dopamine transporter (DAT) showed a similar reproducibility over a five year period (DVR=2.17{+-}0.39, N=52), again irrespective of radionuclide or sex. These studies demonstrate that use of a consistent in vivo technique can provide reliable measures of specific binding of radioligands to high affinity sites in the rat brain.

  20. Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324.

    Directory of Open Access Journals (Sweden)

    Mitun Chakraborty

    Full Text Available The alcohol oxidase (AOx cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g-1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter and catalytic efficiency (kcat/Km of 7829.5 min-1 mM-1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.

  1. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  2. Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

    Directory of Open Access Journals (Sweden)

    Smith Eric L

    2008-01-01

    Full Text Available Abstract Background The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1, and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3. PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members. Results We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind in vivo. Conclusion These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1

  3. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  4. High-Affinity Quasi-Specific Sites in the Genome: How the DNA-Binding Proteins Cope with Them

    Science.gov (United States)

    Chakrabarti, J.; Chandra, Navin; Raha, Paromita; Roy, Siddhartha

    2011-01-01

    Many prokaryotic transcription factors home in on one or a few target sites in the presence of a huge number of nonspecific sites. Our analysis of λ-repressor in the Escherichia coli genome based on single basepair substitution experiments shows the presence of hundreds of sites having binding energy within 3 Kcal/mole of the OR1 binding energy, and thousands of sites with binding energy above the nonspecific binding energy. The effect of such sites on DNA-based processes has not been fully explored. The presence of such sites dramatically lowers the occupation probability of the specific site far more than if the genome were composed of nonspecific sites only. Our Brownian dynamics studies show that the presence of quasi-specific sites results in very significant kinetic effects as well. In contrast to λ-repressor, the E. coli genome has orders of magnitude lower quasi-specific sites for GalR, an integral transcription factor, thus causing little competition for the specific site. We propose that GalR and perhaps repressors of the same family have evolved binding modes that lead to much smaller numbers of quasi-specific sites to remove the untoward effects of genomic DNA. PMID:21889449

  5. Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

    Directory of Open Access Journals (Sweden)

    Michael Allevato

    Full Text Available The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX bind Enhancer box (E-box DNA elements (CANNTG and have the greatest affinity for the canonical MYC E-box (CME CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87% of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

  6. The party effect: Prediction of future alcohol use based on exposure to specific alcohol advertising content

    Science.gov (United States)

    Morgenstern, Matthis; Li, Zhongze; Li, Zhigang; Sargent, James D.

    2016-01-01

    Aims To test whether exposure to party-related alcohol advertising is associated with drinking behavior in a national US sample of adolescents and young adults, independently of exposure to other alcohol advertising. Design Longitudinal telephone- and web-based surveys conducted in 2011 and 2013. Setting All regions of the United States, participants selected via mixed-mode random-digit-dial landline and cellphone frames. Participants A sample of 2541 respondents with a mean age of 18.1 years (51.6% female) of which 1053 (41%) never had a whole drink of alcohol and 1727 (67%) never had six or more drinks during one drinking occasion. Measurements Outcome measures were onset of alcohol use and binge drinking during the study interval. Primary predictor was exposure to television alcohol advertising, operationalized as contact frequency and brand recall for 20 randomly selected alcohol advertisements. Independent post-hoc analyses classified all ads as “party” or “non-party” ads. Sociodemographics, sensation seeking, alcohol expectancies, and alcohol use of friends and family were assessed as covariates. Findings Onset rates for having the first whole drink of alcohol and for first binge drinking were 49.2% and 29.5%, respectively. On average, about half (M = 10.2) of the 20 alcohol advertisements in each individual survey were “party” ads. If both types of exposures (“party” and “non-party”) were included in the regression model, only “party” exposure remained a significant predictor of alcohol use onset (AOR=19.17; 95%CI 3.72–98.79) and binge drinking onset (AOR=3.87; 95%CI 1.07–13.99) after covariate control. Conclusions Adolescents and young adults with higher exposure to alcohol advertisements using a partying theme had higher rates of alcohol use and binge drinking onset, even after control of exposure to other types of alcohol advertisements. PMID:27343140

  7. Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

    Science.gov (United States)

    Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

    2018-05-01

    The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

  8. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Science.gov (United States)

    Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  9. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  10. Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections

    International Nuclear Information System (INIS)

    Naidu, S.S.; Erdei, J.; Forsgren, A.; Naidu, A.S.; Czirok, E.; Gado, I.; Kalfas, S.; Thoren, A.

    1991-01-01

    The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125 I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binging EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125 I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subephithelial matrix proteins and carbohydrates tested (in 10 4 -fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125 I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (K a ) of 1.4 x 10 -7 M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli. (au)

  11. Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins

    Directory of Open Access Journals (Sweden)

    Vergauwen Bjorn

    2011-11-01

    Full Text Available Abstract Background The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp permease of Escherichia coli. The solute binding protein (SBP that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA, and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences. Results Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal

  12. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  13. Behavioral neuroadaptation to alcohol : from glucocorticoids to histone acetylation

    Directory of Open Access Journals (Sweden)

    Daniel Beracochea

    2016-10-01

    Full Text Available A prime mechanism that contributes to the development and maintenance of alcoholism is the dysregulation of the hypothalamic-pituitary-adrenal (HPA axis activity and the release of glucocorticoids (cortisol in humans and primates, corticosterone in rodents from the adrenal glands. In the brain, sustained, local elevation of glucocorticoid concentration even long after cessation of chronic alcohol consumption compromises functional integrity of a circuit including the prefrontal cortex, the hippocampus and the amygdala. These structures are implicated in learning and memory processes as well as in orchestrating neuroadaptive responses to stress and anxiety responses. Thus, potentiation of anxiety-related neuroadaptation by alcohol is characterized by an abnormally amygdala hyperactivity coupled with a hypofunction of the prefrontal cortex and the hippocampus. This review describes research on molecular and epigenetic mechanisms by which alcohol causes distinct region-specific adaptive changes in gene expression patterns and ultimately, leads to a variety of cognitive and behavioral impairments on prefrontal- and hippocampal-based tasks. Alcohol-induced neuroadaptations involve the dysregulation of numerous signaling cascades, leading to long-term changes in transcriptional profiles of genes, through the actions of transcription factors such as CREB (cAMP response element binding protein and chromatin remodeling due to post-translational modifications of histone proteins. We describe the role of prefrontal-hippocampus-amygdala circuit in mediating the effects of acute and chronic alcohol on learning and memory, and region-specific molecular and epigenetic mechanisms involved in this process. This review first discusses the importance of brain region-specific dysregulation of glucocorticoid concentration in the development of alcohol dependence and describes on how persistently increased glucocorticoid levels in prefrontal cortex may be involved in

  14. Measurement of specific [3H]-ouabain binding to different types of human leucocytes

    DEFF Research Database (Denmark)

    Boon, Arnold; Oh, V M; Taylor, John E.

    1984-01-01

    We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature...... were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different...

  15. Effects of Specific Alcohol Control Policy Measures on Alcohol-Related Mortality in Russia from 1998 to 2013.

    Science.gov (United States)

    Khaltourina, Daria; Korotayev, Andrey

    2015-09-01

    To elucidate the possible effects of alcohol control policy measures on alcohol-related mortality in Russia between 1998 and 2013. Trends in mortality, alcohol production and sales were analyzed in conjunction with alcohol control legislative measures. Correlation analysis of health and alcohol market indicators was performed. Ethyl alcohol production was the strongest correlate of alcohol-related mortality, which is probably due to the fact that ethyl alcohol is used for both recorded and unrecorded alcohol production. Measures producing greatest mortality reduction effect included provisions which reduced ethyl alcohol production (introduction of minimum authorized capital for ethyl alcohol and liquor producers in 2006 and the requirement for distillery dreg processing), as well as measures to tax and denaturize ethanol-containing liquids in 2006. Liquor tax decrease in real terms was associated with rising mortality in 1998-1999, while excise tax increase was associated with mortality reduction in 2004 and since 2012. Conventional alcohol control measures may also have played a moderately positive role. Countries with high alcohol-related mortality should aim for a reduction in spirits consumption as a major health policy. Alcohol market centralization and reduction of the number of producers can have immediate strong effects on mortality. These measures should be combined with an increase in alcohol taxes and prices, as well as other established alcohol policy measures. In 2015 in Russia, this is not being implemented. In Russia, legislation enforcement including excise tax collection remains the major challenge. Another challenge will be the integration into the Eurasian Economic Union. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  16. The party effect: prediction of future alcohol use based on exposure to specific alcohol advertising content.

    Science.gov (United States)

    Morgenstern, Matthis; Li, Zhongze; Li, Zhigang; Sargent, James D

    2017-01-01

    To test whether exposure to party-related alcohol advertising is associated with drinking behavior in a national US sample of adolescents and young adults, independently of exposure to other alcohol advertising. Longitudinal telephone- and web-based surveys conducted in 2011 and 2013. All regions of the United States, participants selected via mixed-mode random-digit-dial landline and cellphone frames. A sample of 705 respondents who never had a whole drink of alcohol at baseline (mean age 16.9 years, 53.3% female) and a sample of 1036 who never had six or more drinks during one drinking occasion (mean age 17.4 years, 55.8% female). Outcome measures were onset of alcohol use and binge drinking during the study interval. Primary predictor was exposure to television alcohol advertising, operationalized as contact frequency and brand recall for 20 randomly selected alcohol advertisements. Independent post-hoc analyses classified all advertisements as 'party' or 'non-party' advertisements. Socio-demographics, sensation-seeking, alcohol expectancies and alcohol use of friends and family were assessed as covariates. Onset rates for having the first whole drink of alcohol and for first binge drinking were 49.2% and 29.5%, respectively. On average, approximately half (median = 10.2) of the 20 alcohol advertisements in each individual survey were 'party' advertisements. If both types of exposures ('party' and 'non-party') were included in the regression model, only 'party' exposure remained a significant predictor of alcohol use onset [adjusted odds ratio (AOR) = 19.17; 95% confidence interval (CI) = 3.72-98.79] and binge drinking onset (AOR = 3.87; 95% CI = 1.07-13.99) after covariate control. Adolescents and young adults in the United States appear to have higher rates of alcohol use and binge drinking onset if they have higher exposure to alcohol advertisements using a partying theme, independently of the amount of exposure to alcohol advertisements with non

  17. Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

    Science.gov (United States)

    Trudell, James R.; Harris, R. Adron

    2014-01-01

    Alcohols and other anesthetic agents dramatically alter neurologic function in a wide range of organisms, yet their molecular sites of action remain poorly characterized. Pentameric ligand-gated ion channels, long implicated in important direct effects of alcohol and anesthetic binding, have recently been illuminated in renewed detail thanks to the determination of atomic-resolution structures of several family members from lower organisms. These structures provide valuable models for understanding and developing anesthetic agents and for allosteric modulation in general. This review surveys progress in this field from function to structure and back again, outlining early evidence for relevant modulation of pentameric ligand-gated ion channels and the development of early structural models for ion channel function and modulation. We highlight insights and challenges provided by recent crystal structures and resulting simulations, as well as opportunities for translation of these newly detailed models back to behavior and therapy. PMID:24515646

  18. Specific binding component of the 'inactive' stereoisomer (S,S)-[125I]IQNB to rat brain muscarinic receptors in vivo

    International Nuclear Information System (INIS)

    Boulay, Sheila F.; McRee, R. Carter; Cohen, Victor I.; Sood, Virendar K.; Zeeberg, Barry R.; Reba, Richard C.

    1996-01-01

    In vivo nonspecific binding can be estimated using the inactive stereoisomer of a receptor radioligand. However, the binding of the inactive stereoisomer may be partially specific. Specific binding of the inactive (S,S)-[ 125 I]IQNB was estimated from the inhibition induced by a competing nonradioactive ligand. This technique differed from the usual approach, since it was used to study the inactive rather than the active stereoisomer. The results indicate that there is substantial specific binding for (S,S)-[ 125 I]IQNB

  19. Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

    International Nuclear Information System (INIS)

    De Weck, Z.; Pande, J.; Kaegi, J.H.R.

    1987-01-01

    Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change as measured by exchange retardation is considerably larger for the NAD + than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD + -pyrazole, PHLDH-NAD + -oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD + is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change

  20. What are other parents saying? Perceived parental communication norms and the relationship between alcohol-specific parental communication and college student drinking

    Science.gov (United States)

    Napper, Lucy E.; Hummer, Justin F.; Lac, Andrew; LaBrie, Joseph W.

    2013-01-01

    This study examined parents’ normative perceptions of other college parents’ alcohol-specific communication, and how parents’ perceived communication norms and alcohol-specific communication relate to student drinking outcomes. A sample of 457 student-parent dyads were recruited from a mid-size university. Students completed web-based assessments of alcohol-related attitudes and behaviors. Parents completed alcohol-specific measures of communication norms and parent-child communication, including communication content (i.e., targeted communication) and frequency of communication. Results indicated that parents overestimated how much other parents talked to their college students about the frequency and quantity of alcohol use, but underestimated how often parents initiated conversations about alcohol. In a path model, perceived communication norms positively predicted both targeted communication and frequency of communication. Perceived communication norms and targeted communication negatively predicted students’ attitude toward alcohol use. In contrast, more frequent communication predicted students holding more approving attitudes toward alcohol. The relationship between parents’ perceived communication norms and students’ drinking behaviors was mediated by the parental communication variables and student attitudes. Tests of indirect effects were undertaken to examine meditational processes. The findings underscore relations involving parental perceived communication norms and parents’ own alcohol communication and their children’s drinking outcomes. The complex relationships of different types of parental communication and student outcomes warrant further research. PMID:24128293

  1. Specific binding of large aggregates of amphiphilic molecules to the respective antibodies.

    Science.gov (United States)

    Nabok, Alexei; Tsargorodskaya, Anna; Holloway, Alan; Starodub, Nikolay F; Demchenko, Anna

    2007-07-31

    The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.

  2. Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

    Science.gov (United States)

    D’Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; Erasmus, M. Frank; Hraber, Peter; Bradbury, Andrew R. M.

    2018-01-01

    Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with the same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding. PMID:29568296

  3. Ascorbic acid prevents nonreceptor specific binding of [3H]-5-hydroxytryptamine to bovine cerebral cortex membranes

    International Nuclear Information System (INIS)

    Hamblin, M.W.; Adriaenssens, P.I.; Ariani, K.; Cawthon, R.M.; Stratford, C.A.; Tan, G.L.; Ciaranello, R.D.

    1987-01-01

    [ 3 H]-5-Hydroxytryptamine ([ 3 H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics ''specific'' ligand/receptor binding in that it is inhibited by 10 microM unlabeled 5-HT. This inhibition is not competitive, but rather is due to the prevention of [ 3 H]-5-HT breakdown by excess unlabeled 5-HT. Unlike genuine ligand/receptor binding, the binding of [ 3 H]-5-HT breakdown products is essentially irreversible and does not display a tissue distribution consistent with binding to authentic 5-HT receptors. [ 3 H]-5-HT decomposition can be eliminated by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible [ 3 H]-5-HT binding. When [ 3 H] 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain membranes themselves affords protection against [ 3 H]-5-HT degradation equal to ascorbic acid. This protection is effective below final [ 3 H]-5-HT concentrations of 10 nM. Above 10 nM [ 3 H]-5-HT, addition of ascorbic acid or other antioxidants is necessary to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless irreversible. When care is taken to limit [ 3 H]-5-HT oxidation, the only reversible and saturable specific binding sites observed are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of low affinity (Kd = 15-250 nM) [ 3 H]-5-HT binding sites in brain membrane preparations

  4. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  5. In Vivo Imaging of Cerebral Dopamine D3 Receptors in Alcoholism

    Science.gov (United States)

    Erritzoe, David; Tziortzi, Andri; Bargiela, David; Colasanti, Alessandro; Searle, Graham E; Gunn, Roger N; Beaver, John D; Waldman, Adam; Nutt, David J; Bani, Massimo; Merlo-Pich, Emilio; Rabiner, Eugenii A; Lingford-Hughes, Anne

    2014-01-01

    Animal studies support the role of the dopamine D3 receptor (DRD3) in alcohol reinforcement or liking. Sustained voluntary alcohol drinking in rats has been associated with an upregulation of striatal DRD3 gene expression and selective blockade of DRD3 reduces ethanol preference, consumption, and cue-induced reinstatement. In vivo measurement of DRD3 in the living human brain has not been possible until recently owing to a lack of suitable tools. In this study, DRD3 status was assessed for the first time in human alcohol addiction. Brain DRD3 availability was compared between 16 male abstinent alcohol-dependent patients and 13 healthy non-dependent age-matched males using the DRD3-preferring agonist positron emission tomography (PET) radioligand [11C]PHNO with and without blockade with a selective DRD3 antagonist (GSK598809 60 mg p.o.). In striatal regions of interest, where the [11C]PHNO PET signal represents primarily DRD2 binding, no differences were seen in [11C]PHNO binding between the groups at baseline. However, baseline [11C]PHNO binding was higher in alcohol-dependent patients in hypothalamus (VT: 16.5±4 vs 13.7±2.9, p=0.040), a region in which the [11C]PHNO signal almost entirely reflects DRD3 availability. The reductions in regional receptor binding (VT) following a single oral dose of GSK598809 (60 mg) were consistent with those observed in previous studies across all regions. There were no differences in regional changes in VT following DRD3 blockade between the two groups, indicating that the regional fractions of DRD3 are similar in the two groups, and the increased [11C]PHNO binding in the hypothalamus in alcohol-dependent patients is explained by elevated DRD3 in this group. Although we found no difference between alcohol-dependent patients and controls in striatal DRD3 levels, increased DRD3 binding in the hypothalamus of alcohol-dependent patients was observed. This may be relevant to the development of future therapeutic strategies to

  6. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-β peptide

    International Nuclear Information System (INIS)

    Ghalebani, Leila; Wahlström, Anna; Danielsson, Jens; Wärmländer, Sebastian K.T.S.; Gräslund, Astrid

    2012-01-01

    Highlights: ► Cu(II) and Zn(II) display pH-dependent binding to the Aβ(1–40) peptide. ► At pH 7.4 both metal ions display residue-specific binding to the Aβ peptide. ► At pH 5.5 the binding specificity is lost for Zn(II). ► Differential Cu(II) and Zn(II) binding may help explain metal-induced AD toxicity. -- Abstract: Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer’s disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with 15 N- and 13 C, 15 N-labeled Aβ(1–40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.

  7. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  8. 27 CFR 30.66 - Table 6, showing respective volumes of alcohol and water and the specific gravity in both air and...

    Science.gov (United States)

    2010-04-01

    ... respective volumes of alcohol and water and the specific gravity in both air and vacuum of spirituous liquor... volumes of alcohol and water and the specific gravity in both air and vacuum of spirituous liquor. This... gallon of water in air by the specific gravity in air of the spirits—8.32823 by 0.88862—the product (7...

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

    Science.gov (United States)

    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.

  10. Specific DNA-binding proteins and DNA sequences involved in steroid hormone regulation of gene expression

    International Nuclear Information System (INIS)

    Spelsberg, T.; Hora, J.; Horton, M.; Goldberger, A.; Littlefield, B.; Seelke, R.; Toyoda, H.

    1987-01-01

    Steroid hormones circulate in the blood and are taken by target cells via complexes with intracellular binding proteins termed receptors, that are hormone and tissue specific. Each receptor binds it specific steroid with very high affinity, having an equilibrium dissociation constant (K/sub d/) in the range of 10 -9 to 10 -10 M. Once bound by their specific steroid hormones, the steroid receptors undergo a conformational change which allows them to bind with high affinity to sites on chromatin, termed nuclear acceptor sites. There are estimated 5,000 to 10,000 of these sites expressed with an equal number not expressed (''masked'') in intact chromatin. The result of the binding to nuclear acceptor sites is an alteration of gene transcription or, in some cases, gene expression as measured by the changing levels of specific RNAs and proteins in that target tissue. Each steroid regulates specific effects on the RNA and protein profiles. The chronology of the above mechanism of action after injection of radiolabelled steroid as is follows: Steroid-receptor complex formation (1 minute), nuclear acceptor sites (2 minutes), effects on RNA synthesis (10 to 30 minutes), and finally the changing protein profiles via changes in protein synthesis and protein turnover (1 to 6 hours). Thus steroid receptors represent one of the first identified intracellular gene regulation proteins. The receptor molecules themselves are regulated by the presence or absence of the steroid molecule

  11. Regulation of Alcohol Extinction and Cue-Induced Reinstatement by Specific Projections among Medial Prefrontal Cortex, Nucleus Accumbens, and Basolateral Amygdala.

    Science.gov (United States)

    Keistler, Colby R; Hammarlund, Emma; Barker, Jacqueline M; Bond, Colin W; DiLeone, Ralph J; Pittenger, Christopher; Taylor, Jane R

    2017-04-26

    The ability to inhibit drinking is a significant challenge for recovering alcoholics, especially in the presence of alcohol-associated cues. Previous studies have demonstrated that the regulation of cue-guided alcohol seeking is mediated by the basolateral amygdala (BLA), nucleus accumbens (NAc), and medial prefrontal cortex (mPFC). However, given the high interconnectivity between these structures, it is unclear how mPFC projections to each subcortical structure, as well as projections between BLA and NAc, mediate alcohol-seeking behaviors. Here, we evaluate how cortico-striatal, cortico-amygdalar, and amygdalo-striatal projections control extinction and relapse in a rat model of alcohol seeking. Specifically, we used a combinatorial viral technique to express diphtheria toxin receptors in specific neuron populations based on their projection targets. We then used this strategy to create directionally selective ablations of three distinct pathways after acquisition of ethanol self-administration but before extinction and reinstatement. We demonstrate that ablation of mPFC neurons projecting to NAc, but not BLA, blocks cue-induced reinstatement of alcohol seeking and neither pathway is necessary for extinction of responding. Further, we show that ablating BLA neurons that project to NAc disrupts extinction of alcohol approach behaviors and attenuates reinstatement. Together, these data provide evidence that the mPFC→NAc pathway is necessary for cue-induced reinstatement of alcohol seeking, expand our understanding of how the BLA→NAc pathway regulates alcohol behavior, and introduce a new methodology for the manipulation of target-specific neural projections. SIGNIFICANCE STATEMENT The vast majority of recovering alcoholics will relapse at least once and understanding how the brain regulates relapse will be key to developing more effective behavior and pharmacological therapies for alcoholism. Given the high interconnectivity of cortical, striatal, and limbic

  12. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  13. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  14. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    Science.gov (United States)

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  15. Do pre-drinkers consume more alcohol than non-pre-drinkers on an event-specific night out?

    DEFF Research Database (Denmark)

    Østergaard, Jeanette; Skov, Peter Rohde

    2014-01-01

    Introduction and Aims Young people drinking heavily before going out to bars and clubs is associated with alcohol-related harm and therefore of great public concern. This study examines whether pre-drinkers consume more alcohol than non-pre-drinkers on an event-specific night out in England...... demographics, socioeconomic status, frequency of intoxication and alcohol unit intake before and during the young people's night out. Results A mixed linear model performed on the panel mobile survey shows that pre-drinkers in England and Denmark consume 9.185 (P ..., respectively, more than the non-pre-drinkers. However, in both countries pre-drinkers consume 3.430 (P alcohol on-premises than the non-pre-drinkers. Discussion and Conclusion Pre-drinking is a widespread phenomenon in England and Denmark, with more than half of young...

  16. Age-Specific Associations Between Violence Exposure and Past 30-Day Marijuana and Alcohol Use.

    Science.gov (United States)

    Goldstick, Jason E; Heinze, Justin E; Stoddard, Sarah A; Cunningham, Rebecca M; Zimmerman, Marc A

    2018-04-23

    Using data from a cohort study of students at risk for high school dropout, we examined associations between violence exposure and past 30-day alcohol and marijuana use. We used varying-coefficient regression with person-level fixed effects to estimate how those associations changed within-person across ages approximately 14-23. Generally, violence perpetration was most strongly associated with substance use, within-person. Substance use became increasingly associated with both observed violence and violence perpetration during early/middle adolescence; this increase continued longer into development (age 18+) for alcohol use. Across most of the age range studied here, violence victimization was minimally associated with within-person changes in substance use. Results indicate age-specific associations between violence exposure and alcohol and other drug use, which may be useful for informing prevention strategies. © 2018 Society for Research on Adolescence.

  17. Deep convolutional neural networks for pan-specific peptide-MHC class I binding prediction.

    Science.gov (United States)

    Han, Youngmahn; Kim, Dongsup

    2017-12-28

    Computational scanning of peptide candidates that bind to a specific major histocompatibility complex (MHC) can speed up the peptide-based vaccine development process and therefore various methods are being actively developed. Recently, machine-learning-based methods have generated successful results by training large amounts of experimental data. However, many machine learning-based methods are generally less sensitive in recognizing locally-clustered interactions, which can synergistically stabilize peptide binding. Deep convolutional neural network (DCNN) is a deep learning method inspired by visual recognition process of animal brain and it is known to be able to capture meaningful local patterns from 2D images. Once the peptide-MHC interactions can be encoded into image-like array(ILA) data, DCNN can be employed to build a predictive model for peptide-MHC binding prediction. In this study, we demonstrated that DCNN is able to not only reliably predict peptide-MHC binding, but also sensitively detect locally-clustered interactions. Nonapeptide-HLA-A and -B binding data were encoded into ILA data. A DCNN, as a pan-specific prediction model, was trained on the ILA data. The DCNN showed higher performance than other prediction tools for the latest benchmark datasets, which consist of 43 datasets for 15 HLA-A alleles and 25 datasets for 10 HLA-B alleles. In particular, the DCNN outperformed other tools for alleles belonging to the HLA-A3 supertype. The F1 scores of the DCNN were 0.86, 0.94, and 0.67 for HLA-A*31:01, HLA-A*03:01, and HLA-A*68:01 alleles, respectively, which were significantly higher than those of other tools. We found that the DCNN was able to recognize locally-clustered interactions that could synergistically stabilize peptide binding. We developed ConvMHC, a web server to provide user-friendly web interfaces for peptide-MHC class I binding predictions using the DCNN. ConvMHC web server can be accessible via http://jumong.kaist.ac.kr:8080/convmhc

  18. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    International Nuclear Information System (INIS)

    Öberg, Christine; Belikov, Sergey

    2012-01-01

    Highlights: ► wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, ΔN-hH1.4, were compared. ► Both histones bind to chromatin, however, ΔN-hH1.4 displays lower binding affinity. ► Interaction of ΔN-hH1.4 with chromatin includes a significant unspecific component. ► N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). The ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that ΔN-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  19. Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

    Science.gov (United States)

    Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2016-03-14

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  20. The gender specific mediational pathways between parenting styles, neuroticism, pathological reasons for drinking, and alcohol-related problems in emerging adulthood.

    Science.gov (United States)

    Patock-Peckham, Julie A; Morgan-Lopez, Antonio A

    2009-03-01

    Mediational links between parenting styles, neuroticism, pathological reasons for drinking, alcohol use and alcohol-related problems were tested. A two-group SEM path model with 441 (216 female, 225 male) college students was examined. In general, pathological reasons for drinking mediated the impact of neuroticism on alcohol use and alcohol-related problems. A different pattern of relationships was found for each of the two genders. Perceptions of having an authoritarian father were positively linked to higher levels of neuroticism among males but this pattern was not found among females. For males, neuroticism mediated the impact of having an authoritarian father on pathological reasons for drinking with pathological reasons for drinking mediating the impact of neuroticism on alcohol-related problems. Perceptions of having a permissive father were linked to lower levels of neuroticism in females (but have been found as a consistent risk factor for other pathways to alcohol use elsewhere). Compared with other work in this area, these findings indicate parental influences regarding vulnerabilities for alcohol use may be specific to parent-child gender matches for some pathways and specific to one parent (irrespective of child gender) for other pathways.

  1. Gender-specific mediational links between parenting styles, parental monitoring, impulsiveness, drinking control, and alcohol-related problems.

    Science.gov (United States)

    Patock-Peckham, Julie A; King, Kevin M; Morgan-Lopez, Antonio A; Ulloa, Emilio C; Moses, Jennifer M Filson

    2011-03-01

    Recently, it has been suggested that traits may dynamically change as conditions change. One possible mechanism that may influence impulsiveness is parental monitoring. Parental monitoring reflects a knowledge regarding one's offspring's whereabouts and social connections. The aim of this investigation was to examine potential gender-specific parental influences to impulsiveness (general behavioral control), control over one's own drinking (specific behavioral control), and alcohol-related problems among individuals in a period of emerging adulthood. Direct and mediational links between parenting styles (permissive, authoritarian, and authoritative), parental monitoring, impulsiveness, drinking control, and alcohol-related problems were investigated. A multiple-group, SEM model with (316 women, 265 men) university students was examined. In general, the overall pattern among male and female respondents was distinct. For daughters, perceptions of a permissive father were indirectly linked to more alcohol-related problems through lower levels of monitoring by fathers and more impulsive symptoms. Perceptions of an authoritative father were also indirectly linked to fewer impulsive symptoms through higher levels of monitoring by fathers among daughters. For men, perceptions of a permissive mother were indirectly linked to more alcohol-related problems through lower levels of monitoring by mothers and more impulsive symptoms. For sons, perceptions of mother authoritativeness were indirectly linked to fewer alcohol-related problems through more monitoring by mothers and fewer impulsive symptoms. Monitoring by an opposite-gender parent mediated the link between parenting styles (i.e., permissive, authoritative) on impulsiveness.

  2. Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

    Science.gov (United States)

    Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan

    2017-07-20

    Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

  3. Different roles suggested by sex-biased expression and pheromone binding affinity among three pheromone binding proteins in the pink rice borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Jin, Jun-Yan; Li, Zhao-Qun; Zhang, Ya-Nan; Liu, Nai-Yong; Dong, Shuang-Lin

    2014-07-01

    Pheromone binding proteins (PBPs) are thought to bind and transport hydrophobic sex pheromone molecules across the aqueous sensillar lymph to specific pheromone receptors on the dendritic membrane of olfactory neurons. A maximum of 3 PBP genes have been consistently identified in noctuid species, and each of them shares high identity with its counterparts in other species within the family. The functionality differences of the 3 proteins are poorly understood. In the present study, 3 PBP cDNAs (SinfPBP1, 2, 3) were identified from the pink rice borer, Sesamia inferens, for the first time. The quantitative real-time PCR indicated that the 3 PBPs displayed similar temporal but very different sex related expression profiles. Expression of SinfPBP1 and SinfPBP2 were highly and moderately male biased, respectively, while SinfPBP3 was slightly female biased, as SinfPBPs were expressed at very different levels (PBP1>PBP2≫PBP3) in male antennae, but at similar levels in female antennae. Furthermore, the 3 SinfPBPs displayed different ligand binding profiles in fluorescence competitive binding assays. SinfPBP1 exhibited high and similar binding affinities to all 3 sex pheromone components (Ki=0.72-1.60 μM), while SinfPBP2 showed selective binding to the alcohol and aldehyde components (Ki=0.78-1.71 μM), and SinfPBP3 showed no obvious binding to the 3 sex pheromone components. The results suggest that SinfPBP1 plays a major role in the reception of female sex pheromones in S. inferens, while SinfPBP3 plays a least role (if any) and SinfPBP2 functions as a recognizer of alcohol and aldehyde components. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Prognostic value of insulinlike growth factor I and its binding protein in patients with alcohol-induced liver disease. EMALD group

    DEFF Research Database (Denmark)

    Møller, Søren; Becker, Povl Ulrik; Juul, A

    1996-01-01

    Insulinlike growth factor I (IGF-I) is a single-polypeptide chain with important anabolic and endocrine activities. The liver is the major source of IGF-I and its binding protein, IGFBP-3. Circulating concentrations of IGF-I and IGFBP-3 are decreased in patients with chronic liver disease...... and correlate with the severity. The aim of this study was to assess the additional prognostic value of IGF-I and IGFBP-3 in patients entered in a large multicenter study (EMALD). Three hundred thirty-seven patients with alcohol-induced liver disease were studied in a randomized placebo-controlled trial...... of malotilate with a mean follow-up period of 569 days (range, 7-1,544). A multivariate Cox regression analysis of pertinent clinical and biochemical variables showed a significant independent prognostic value of years of alcohol intake, coagulation factors 2, 7, and 10, alkaline phosphatases, serum creatinine...

  5. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  6. 125I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    International Nuclear Information System (INIS)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of 125 I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class A/B diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more 125 I-hEGF than did fetal membranes (P 125 I-hEGF binding to fetal membranes from the various pregnancy states (P 125 I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P 125 I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P 125 I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P 125 I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone. (author)

  7. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Amy L Bauer

    2010-11-01

    Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  8. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J

    2000-01-01

    ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed...... of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence...... in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function....

  9. Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the arterior pituitary gland

    International Nuclear Information System (INIS)

    Guillemette, G.; Balla, T.; Baukal, A.J.; Catt, K.J.

    1987-01-01

    The properties of inositol 1,4,5-trisphosphate (InsP 3 ) receptor sites in the anterior pituitary were evaluated by binding studies with InsP 3 labeled with 32 P to high specific radioactivity. Specific binding of Ins[ 32 P]P 3 was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP 3 binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0 0 C. Scatchard analysis of the binding data was consistent with a single set of high affinity sites. The specificity of Ins[ 32 P]P 3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP 3 binding sites, the Ca 2+ -releasing activity of InsP 3 was measured in pituitary membrane preparations. Under physiological conditions within the cytosol, the high-affinity InsP 3 binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP 3 triggers Ca 2+ mobilization in the anterior pituitary gland

  10. Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT genomic binding patterns discerns cell-specific cis-regulatory modules

    Directory of Open Access Journals (Sweden)

    Kang Keunsoo

    2013-01-01

    Full Text Available Abstract Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM, which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

  11. A Preliminary Investigation of the Effect of Acute Alcohol on Dopamine Transmission as Assessed by [11 C]-(+)-PHNO.

    Science.gov (United States)

    Thiruchselvam, Thulasi; Wilson, Alan A; Boileau, Isabelle; Le Foll, Bernard

    2017-06-01

    Previous positron emission tomography (PET) studies exploring the effect of acute alcohol on dopamine (DA) levels have yielded inconsistent results, with only some studies suggesting increased synaptic DA levels after an alcohol challenge. The D 2 /D 3 agonist radiotracer, [ 11 C]-(+)-propyl-hexahydro-naphtho-oxazin ([ 11 C]-(+)-PHNO), has greater sensitivity to synaptic DA fluctuation than previously used antagonist radiotracers and is in principle more suitable for imaging alcohol-induced changes in DA. Its high affinity for the D 3 receptor also enables measuring changes in D 3 -rich brain areas which have previously been unexplored. The aim of this study was to investigate whether alcohol reduces [ 11 C]-(+)-PHNO binding in the striatum and in D 3 -rich extra-striatal areas. Eight healthy drinkers underwent 2 [ 11 C]-(+)-PHNO PET scans following alcohol and placebo in a randomized, single-blind, crossover design. [ 11 C]-(+)-PHNO binding in the striatum and in the extra-striatal regions were compared between the 2 scans. Acute alcohol administration did not significantly reduce [ 11 C]-(+)-PHNO binding in either the limbic striatum (d = 0.64), associative striatum (d < 0.20), or the sensorimotor striatum (d < 0.15). Similarly, there were no changes in binding in the D 3 -rich areas of the ventral pallidum (d = 0.53), substantia nigra (d < 0.15), or globus pallidus (d < 0.15). However, greater percent change in [ 11 C]-(+)-PHNO binding (ΔBP ND ) between scans was related to lower blood alcohol levels. Using the agonist radiotracer, [ 11 C]-(+)-PHNO, our preliminary findings suggest that alcohol is not associated with robust changes in tracer binding in striatal or extra-striatal regions. However, we found that changes in [ 11 C]-(+)-PHNO binding following alcohol are dependent on blood alcohol levels suggesting that increases in DA may occur at lower stimulating doses. The effect of lower doses of alcohol on DA warrants further investigation in a

  12. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.

    Science.gov (United States)

    Gilbert, G E; Drinkwater, D

    1993-09-21

    Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance.

  13. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Daniil M Prigozhin

    Full Text Available Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

  14. Brand-Specific Consumption of Flavored Alcoholic Beverages among Underage Youth in the United States

    Science.gov (United States)

    Fortunato, Erin K.; Siegel, Michael; Ramirez, Rebecca L.; Ross, Craig; DeJong, William; Albers, Alison B.; Jernigan, David H.

    2013-01-01

    Background Although several studies have identified flavored alcoholic beverages (FABs) as being popular among underage drinkers, no previous study has ascertained the prevalence of brand-specific FAB consumption among a national sample of underage youth. Objectives To ascertain the brand-specific consumption prevalence and consumption share of FABs among a national sample of underage drinkers in the United States. Methods In 2012, we conducted an online, self-administered survey of a national sample of 1,031 underage drinkers, ages 13-20, to determine the prevalence of past 30-day consumption for each of 898 alcoholic beverage brands, including 62 FABs, and each brand’s youth consumption share, based on the estimated total number of standard drinks consumed. There were three brand-specific outcome measures: prevalence of consumption, prevalence of consumption during heavy episodic drinking, and consumption share, defined as the percentage of the total drinks consumed by all respondents combined that was attributable to a particular brand. Results The FAB brands with the highest prevalence of past 30-day consumption were Smirnoff Malt Beverages, 17.7%; Mike’s, 10.8%; Bacardi Malt Beverages, 8.0%; and Four Loko/Four MaXed, 6.1%. Just five brands accounted for almost half (49.1%) of the total consumption share by volume within the FAB category. Conclusion Flavored alcoholic beverages are highly popular among underage drinkers, and their FAB brand preferences are highly concentrated among a small number of brands. To decrease the consumption of FABs by underage youth, all states should re-classify these beverages as distilled spirits rather than beer. PMID:24266600

  15. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

    Directory of Open Access Journals (Sweden)

    Raymond Wilson

    2010-09-01

    and metacyclic forms. Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.

  16. Friendship Context Matters: Examining the Domain Specificity of Alcohol and Depression Socialization Among Adolescents

    NARCIS (Netherlands)

    Giletta, M.; Scholte, R.H.J.; Prinstein, M.J.; Engels, R.C.M.E.; Rabaglietti, E.; Burk, W.J.

    2012-01-01

    Driven by existing socialization theories, this study describes specific friendship contexts in which peer influence of alcohol misuse and depressive symptoms occurs. In the fall and spring of the school year, surveys were administered to 704 Italian adolescents (53 % male, M (age) = 15.53) enrolled

  17. In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

    Science.gov (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

    2012-01-01

    One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

  18. In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

    Directory of Open Access Journals (Sweden)

    Yasaman Ardeshirpour

    Full Text Available One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

  19. Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Keehyuk; Plapp, Bryce V. (Iowa)

    2017-10-01

    The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that

  20. Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93.

    Science.gov (United States)

    Kim, Keehyuk; Plapp, Bryce V

    2017-10-01

    The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/K m ) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD + and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/K m ) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/K m for (S)-2-alcohols without changing V/K m for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme

  1. Sequence specificity and biological consequences of drugs that bind covalently in the minor groove of DNA

    International Nuclear Information System (INIS)

    Hurley, L.H.; Needham-VanDevanter, D.R.

    1986-01-01

    DNA ligands which bind within the minor groove of DNA exhibit varying degrees of sequence selectivity. Factors which contribute to nucleotide sequence recognition by minor groove ligands have been extensively investigated. Electrostatic interactions, ligand and DNA dehydration energies, hydrophobic interactions and steric factors all play significant roles in sequence selectivity in the minor groove. Interestingly, ligand recognition of nucleotide sequence in the minor groove does not involve significant hydrogen bonding. This is in sharp contrast to cellular enzyme and protein recognition of nucleotide sequence, which is achieved in the major groove via specific hydrogen bond formation between individual bases and the ligand. The ability to read nucleotide sequence via hydrogen bonding allows precise binding of proteins to specific DNA sequences. Minor groove ligands examined to date exhibit a much lower sequence specificity, generally binding to a subset of possible sequences, rather than a single sequence. 19 refs., 7 figs

  2. Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol*

    Science.gov (United States)

    Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

    2015-01-01

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

  3. Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for prostate specific antigen binding.

    Science.gov (United States)

    Idil, Neslihan; Perçin, Işık; Karakoç, Veyis; Yavuz, Handan; Aksöz, Nilüfer; Denizli, Adil

    2015-10-01

    The aim of this study was to prepare Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads for prostate specific antigen (PSA) binding and to study binding capacities of the beads using lectin-glycoprotein interactions. Firstly, iron oxide nanoparticles were synthesized by co-precipitation method and then, beads were synthesized by dispersion polymerization in the presence of iron oxide nanoparticles. Con A molecules were both covalently immobilized onto the beads directly and through the spacer arm (1,6-diaminohexane-HDMA). The total PSA and free PSA binding onto the mPGMA-HDMA-Con A beads were higher than that of the mPGMA-Con A beads. Maximum PSA binding capacity was observed as 91.2 ng/g. Approximately 45% of the bound PSA was eluted by using 0.1 M mannose as elution agent. The mPGMA-HDMA-Con A beads could be reused without a remarkable decrease in the binding capacities after 5 binding-desorption cycles. Serum fractions were analyzed using SDS-PAGE. The mPGMA-HDMA-Con A beads could be useful for the detection of PSA and suggested as a model system for other glycoprotein biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Photo-triggered release from liposomes without membrane solubilization, based on binding to poly(vinyl alcohol) carrying a malachite green moiety.

    Science.gov (United States)

    Uda, Ryoko M; Kato, Yutaka; Takei, Michiko

    2016-10-01

    When working with liposomes analogous to cell membranes, it is important to develop substrates that can regulate interactions with the liposome surface in response to light. We achieved a photo-triggered release from liposomes by using a copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG is a neutral polymer under dark conditions, it is photoionized upon exposure to UV light, resulting in the formation of a cationic site for binding to liposomes with a negatively charged surface. Under UV irradiation, PVAMG showed effective interaction with liposomes, releasing the encapsulated compound; however, this release was negligible under dark conditions. The poly(vinyl alcohol) moiety of PVAMG played an important role in the photo-triggered release. This release was caused by membrane destabilization without lipid solubilization. We also investigated different aspects of liposome/PVAMG interactions, including PVAMG-induced fusion between the liposomes and the change in the liposome morphologies. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  6. Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

    2010-01-01

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

  7. Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

    Science.gov (United States)

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

    2010-01-15

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

  8. The influence of duration of follow-up on the association between alcohol and cause-specific mortality in a prospective cohort study

    DEFF Research Database (Denmark)

    Nielsen, Naja Rod; Thygesen, Lau Caspar; Johansen, Ditte

    2005-01-01

    PURPOSE: To investigate whether duration of follow-up influences the association between alcohol and cause-specific mortality in a prospective cohort study with only baseline assessment of alcohol intake. METHODS: In a cohort of 14,223 men and women participating in the first investigation...... of the Copenhagen City Heart Study between 1976 and 1978 and followed until 2001, we assessed whether the association between alcohol and mortality was modified by duration of follow-up. The 24 years of follow-up were divided into four intervals, and Cox survival analyses were conducted separately for these four...... succeeding 6-year periods of follow-up. RESULTS: The authors found differences in the predictive values of alcohol and beverage-specific types of alcohol depending on duration of follow-up both in terms of all-cause mortality and death from coronary heart disease and cancer. The apparent protective effect...

  9. Biodistribution, binding specificity and metabolism of [{sup 18}F]fluoroethylflumazenil in rodents

    Energy Technology Data Exchange (ETDEWEB)

    Leveque, Philippe; Labar, Daniel; Gallez, Bernard E-mail: gallez@cmfa.ucl.ac.be

    2001-10-01

    Pre-clinical studies were carried out in order to characterize in rodents the biodistribution, the binding specificity and the metabolism of [{sup 18}F]Fluoroethylflumazenil ([{sup 18}F]FEF), a potential candidate for in vivo imaging of the benzodiazepine receptors. In vivo competition with flumazenil indicates that [{sup 18}F]FEF binds specifically to the benzodiazepine receptor in the brain. The accumulation of [{sup 18}F]FEF was significantly lower than using [{sup 3}H]Flumazenil. The rather low accumulation in the brain is due to a rapid metabolism of [{sup 18}F]FEF in hydrophylic metabolites which cannot cross the blood brain barrier, and are rapidly eliminated in the urine. Inhibition of the metabolism by acetaminophen (chemically induced hepatitis) led to a significant increase of the radioactivity found in the circulating blood and in the brain, while these results were not observed using classical inhibitors of the cytochrome CYP450, cimetidine and ketoconazole.

  10. The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

    Directory of Open Access Journals (Sweden)

    Bin Qiu

    2016-08-01

    Full Text Available FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1 Fkbp5 KO and wild-type (WT EtOH consumption was tested using a two-bottle choice paradigm; (2 The EtOH elimination rate was measured after intraperitoneal (IP injection of 2.0 g/kg EtOH; (3 Blood alcohol concentration (BAC was measured after 3 h limited access of alcohol; (4 Brain region expression of Fkbp5 was identified using LacZ staining; (5 Baseline corticosterone (CORT was assessed. Additionally, two SNPs, rs1360780 (C/T and rs3800373 (T/G, were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162 from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT. Finally, single nucleotide polymorphisms (SNPs in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

  11. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    Energy Technology Data Exchange (ETDEWEB)

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Lurz, Rudi [Max-Planck Institute for Molecular Genetics, 14195 Berlin (Germany); Calendar, Richard [Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 (United States); Klumpp, Jochen, E-mail: jochen.klumpp@hest.ethz.ch [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Loessner, Martin J. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland)

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  12. Is nutrient intake a gender-specific cause for enhanced susceptibility to alcohol-induced liver disease in women?

    DEFF Research Database (Denmark)

    Wagnerberger, S.; Schäfer, C.; Schwarz, E.

    2008-01-01

    AIM: Women have a higher susceptibility to alcohol-induced liver disease (ALD) than men. Gender-related differences in food preference were described in previous studies for several populations, but not in alcohol abusers. As certain micronutrients are reported to take influence on the development......, the data of calculated daily macro- and micronutrient intake do not suggest any explicit influence of gender-specific nutrition in the development of ALD....

  13. Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

    Directory of Open Access Journals (Sweden)

    McCann Susan E

    2002-05-01

    Full Text Available Abstract Background Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV1 and forced vital capacity (FVC in a random sample of 1555 residents of Western New York, USA. Methods We expressed pulmonary function as percent of predicted normal FEV1 (FEV1% and FVC (FVC% after adjustment for height, age, gender and race. To obtain information on alcohol intake we used a questionnaire that reliably queries total alcohol and beverage specific recent (past 30 days and lifetime alcohol consumption. Results: Using multiple linear regression analysis after adjustment for covariates (pack-years of smoking, weight, smoking status, education, nutritional factors and for FEV1%, in addition, eosinophil count, we observed no significant correlation between total alcohol intake and lung function. However, we found positive associations of recent and lifetime wine intake with FEV1% and FVC%. When we analyzed white and red wine intake separately, the association of lung function with red wine was weaker than for white wine. Conclusion While total alcohol intake was not related to lung function, wine intake showed a positive association with lung function. Although we cannot exclude residual confounding by healthier lifestyle in wine drinkers, differential effects of alcoholic beverages on lung health may exist.

  14. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    Science.gov (United States)

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  15. An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes

    International Nuclear Information System (INIS)

    Evans, T.; Reitman, M.; Felsenfeld, G.

    1988-01-01

    The authors have identified a protein present only in erythroid cells that binds to two adjacent sites within an enhancer region of the chicken β-globin locus. Mutation of the sites, so that binding by the factor can no longer be detected in vitro, leads to a loss of enhancing ability, assayed by transient expression in primary erythrocytes. Binding sites for the erythroid-specific factor (Eryf1) are found within regulatory regions for all chicken globin genes. A strong Eryf1 binding site is also present within the enhancer of at least one human globin gene, and proteins from human erythroid cells (but not HeLa cells) bind to both the chicken and the human sites

  16. Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

    DEFF Research Database (Denmark)

    Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

    2014-01-01

    In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc......-binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (65ZnCl2) blotting technique was optimized to detect zinc-binding prolamins, followed by development of an easy-to-follow nonradioactive colorimetric...... zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

  17. Changes in the α4β2* nicotinic acetylcholine system during chronic controlled alcohol exposure in nonhuman primates.

    Science.gov (United States)

    Hillmer, Ansel T; Tudorascu, Dana L; Wooten, Dustin W; Lao, Patrick J; Barnhart, Todd E; Ahlers, Elizabeth O; Resch, Leslie M; Larson, Julie A; Converse, Alexander K; Moore, Colleen F; Schneider, Mary L; Christian, Bradley T

    2014-05-01

    The precise nature of modifications to the nicotinic acetylcholine receptor (nAChR) system in response to chronic ethanol exposure is poorly understood. The present work used PET imaging to assay α4β2* nAChR binding levels of eight rhesus monkeys before and during controlled chronic ethanol intake. [(18)F]Nifene PET scans were conducted prior to alcohol exposure, and then again after at least 8 months controlled ethanol exposure, including 6 months at 1.5 g/kg/day following a dose escalation period. Receptor binding levels were quantified with binding potentials (BPND) using the cerebellum as a reference region. Alcohol self-administration was assessed as average daily alcohol intake during a 2 month free drinking period immediately following controlled alcohol. Significant decreases in α4β2* nAChR binding were observed in both frontal and insular cortex in response to chronic ethanol exposure. During chronic alcohol exposure, BPND in the lateral geniculate region correlated positively with the amount of alcohol consumed during free drinking. The observed decreases in nAChR availability following chronic alcohol consumption suggest alterations to this receptor system in response to repeated alcohol administration, making this an important target for further study in alcohol abuse and alcohol and nicotine codependence. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  18. NEUROCOGNITIVE ASSESSMENT OF ALCOHOL INPATIENTSDURING RECOVERY FROM ALCOHOLISM*

    Directory of Open Access Journals (Sweden)

    Lilijana Šprah

    2008-05-01

    Our study demonstrated that some alcohol-related cognitive, emotional and motivationaldeficits can also persist to certain extent after several weeks of sobriety. Especially alcoholabstainers with suicidal history revealed a specific neuropsychological profile in this regard. Employed neurocognitive assessment proved as useful approach for clinical evaluation of alcohol abstainers functioning, since cognitive deficits have been also hypothesizedto affect the efficacy of alcoholism treatment

  19. Alcohol's Effects on Lipid Bilayer Properties

    Science.gov (United States)

    Ingólfsson, Helgi I.; Andersen, Olaf S.

    2011-01-01

    Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol's biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1–16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket. PMID:21843475

  20. Alcohol-attributable and alcohol-preventable mortality in Denmark

    DEFF Research Database (Denmark)

    Eliasen, Marie; Becker, Ulrik; Grønbæk, Morten

    2014-01-01

    The aim of the study was to quantify alcohol-attributable and -preventable mortality, totally and stratified on alcohol consumption in Denmark 2010, and to estimate alcohol-related mortality assuming different scenarios of changes in alcohol distribution in the population. We estimated alcohol......-attributable and -preventable fractions based on relative risks of conditions causally associated with alcohol from meta-analyses and information on alcohol consumption in Denmark obtained from 14,458 participants in the Danish National Health Survey 2010 and corrected for adult per capita consumption. Cause-specific mortality...... data were obtained from the Danish Register of Causes of Death. In total, 1,373 deaths among women (5.0 % of all deaths) and 2,522 deaths among men (9.5 % of all deaths) were attributable to alcohol, while an estimated number of 765 (2.8 %) and 583 (2.2 %) deaths were prevented by alcohol...

  1. Background Predictors and Event-Specific Characteristics of Sexual Aggression Incidents: The Roles of Alcohol and Other Factors.

    Science.gov (United States)

    Davis, Kelly Cue; Danube, Cinnamon L; Stappenbeck, Cynthia A; Norris, Jeanette; George, William H

    2015-08-01

    Sexual assault in the United States is an important public health concern. Using prospective longitudinal methods and responses from 217 community men, we examined whether background characteristics predicted subsequent sexual aggression (SA) perpetration during a 3-month follow-up period. We also examined event-specific characteristics of reported SA occurrences. Consistent with predictions, SA perpetration history, aggressive and impulsive personality traits, rape myth attitudes, and alcohol expectancies predicted SA (both non- and alcohol-involved) at follow-up. In addition, alcohol-involved assaults occurred more often with casual (vs. steady) partners but were more likely to involve condom use with casual (vs. steady) partners. Results suggest important avenues for future research and SA prevention efforts. © The Author(s) 2015.

  2. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

    Science.gov (United States)

    Fantl, W J; Escobedo, J A; Martin, G A; Turck, C W; del Rosario, M; McCormick, F; Williams, L T

    1992-05-01

    The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

  3. A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration.

    Science.gov (United States)

    Kuttippurathu, Lakshmi; Patra, Biswanath; Hoek, Jan B; Vadigepalli, Rajanikanth

    2016-03-01

    Liver regeneration after partial hepatectomy is a clinically important process that is impaired by adaptation to chronic alcohol intake. We focused on the initial time points following partial hepatectomy (PHx) to analyze the genome-wide binding activity of NF-κB, a key immediate early regulator. We investigated the effect of chronic alcohol intake on immediate early NF-κB genome-wide localization, in the adapted state as well as in response to partial hepatectomy, using chromatin immunoprecipitation followed by promoter microarray analysis. We found many ethanol-specific NF-κB binding target promoters in the ethanol-adapted state, corresponding to the regulation of biosynthetic processes, oxidation-reduction and apoptosis. Partial hepatectomy induced a diet-independent shift in NF-κB binding loci relative to the transcription start sites. We employed a novel pattern count analysis to exhaustively enumerate and compare the number of promoters corresponding to the temporal binding patterns in ethanol and pair-fed control groups. The highest pattern count corresponded to promoters with NF-κB binding exclusively in the ethanol group at 1 h post PHx. This set was associated with the regulation of cell death, response to oxidative stress, histone modification, mitochondrial function, and metabolic processes. Integration with the global gene expression profiles to identify putative transcriptional consequences of NF-κB binding patterns revealed that several of ethanol-specific 1 h binding targets showed ethanol-specific differential expression through 6 h post PHx. Motif analysis yielded co-incident binding loci for STAT3, AP-1, CREB, C/EBP-β, PPAR-γ and C/EBP-α, likely participating in co-regulatory modules with NF-κB in shaping the immediate early response to PHx. We conclude that adaptation to chronic ethanol intake disrupts the NF-κB promoter binding landscape with consequences for the immediate early gene regulatory response to the acute challenge of PHx.

  4. Fetal Alcohol Syndrome and Fetal Alcohol Effects in Child Development.

    Science.gov (United States)

    Pancratz, Diane R.

    This literature review defines Fetal Alcohol Syndrome (FAS) and Fetal Alcohol Effects (FAE) and considers their causes, diagnoses, prevalence, and educational ramifications. Effects of alcohol during each of the trimesters of pregnancy are summarized. Specific diagnostic characteristics of FAS are listed: (1) growth deficiency, (2) a…

  5. Results of a Two-Year Longitudinal Study of Beverage-Specific Alcohol Use among Adolescents

    Science.gov (United States)

    Moore, Michele Johnson; Werch, Chudley

    2007-01-01

    This study explored beverage-specific alcohol consumption patterns among a sample of high school students over a two-year period. Four hundred fifty-five students completed the validated questionnaire at all three time points (2002, 2003, 2004). Variables of interest included five use measures (past year use, 30-day frequency, quantity, heavy use,…

  6. Alcohol and cause-specific mortality in Russia: a retrospective case-control study of 48,557 adult deaths.

    Science.gov (United States)

    Zaridze, David; Brennan, Paul; Boreham, Jillian; Boroda, Alex; Karpov, Rostislav; Lazarev, Alexander; Konobeevskaya, Irina; Igitov, Vladimir; Terechova, Tatiana; Boffetta, Paolo; Peto, Richard

    2009-06-27

    Alcohol is an important determinant of the high and fluctuating adult mortality rates in Russia, but cause-specific detail is lacking. Our case-control study investigated the effects of alcohol consumption on male and female cause-specific mortality. In three Russian industrial cities with typical 1990s mortality patterns (Tomsk, Barnaul, Biysk), the addresses of 60,416 residents who had died at ages 15-74 years in 1990-2001 were visited in 2001-05. Family members were present for 50,066 decedents; for 48,557 (97%), the family gave proxy information on the decedents' past alcohol use and on potentially confounding factors. Cases (n=43,082) were those certified as dying from causes we judged beforehand might be substantially affected by alcohol or tobacco; controls were the other 5475 decedents. Case versus control relative risks (RRs; calculated as odds ratios by confounder-adjusted logistic regression) were calculated in ever-drinkers, defining the reference category by two criteria: usual weekly consumption always less than 0.5 half-litre bottles of vodka (or equivalent in total alcohol content) and maximum consumption of spirits in 1 day always less than 0.5 half-litre bottles. Other ever-drinkers were classified by usual weekly consumption into three categories: less than one, one to less than three, and three or more (mean 5.4 [SD 1.4]) bottles of vodka or equivalent. In men, the three causes accounting for the most alcohol-associated deaths were accidents and violence (RR 5.94, 95% CI 5.35-6.59, in the highest consumption category), alcohol poisoning (21.68, 17.94-26.20), and acute ischaemic heart disease other than myocardial infarction (3.04, 2.73-3.39), which includes some misclassified alcohol poisoning. There were significant excesses of upper aerodigestive tract cancer (3.48, 2.84-4.27) and liver cancer (2.11, 1.64-2.70). Another five disease groups had RRs of more than 3.00 in the highest alcohol category: tuberculosis (4.14, 3.44-4.98), pneumonia (3

  7. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

    Science.gov (United States)

    Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

    2015-10-30

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  9. DNA binding by the plant-specific NAC transcription factors in crystal and solution

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Lindemose, Søren; Grossmann, J. Günter

    2012-01-01

    angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY...

  10. Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

    International Nuclear Information System (INIS)

    Dold, K.M.; Greenlee, W.F.

    1990-01-01

    A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells

  11. On binding specificity of (6-4) photolyase to a T(6-4)T DNA photoproduct*

    Science.gov (United States)

    Jepsen, Katrine Aalbæk; Solov'yov, Ilia A.

    2017-06-01

    Different factors lead to DNA damage and if it is not repaired in due time, the damaged DNA could initiate mutagenesis and cancer. To avoid this deadly scenario, specific enzymes can scavenge and repair the DNA, but the enzymes have to bind first to the damaged sites. We have investigated this binding for a specific enzyme called (6-4) photolyase, which is capable of repairing certain UV-induced damage in DNA. Through molecular dynamics simulations we describe the binding between photolyase and the DNA and reveal that several charged amino acid residues in the enzyme, such as arginines and lysines turn out to be important. Especially R421 is crucial, as it keeps the DNA strands at the damaged site inside the repair pocket of the enzyme separated. DNA photolyase is structurally highly homologous to a protein called cryptochrome. Both proteins are biologically activated similarly, namely through flavin co-factor photoexcitation. It is, however, striking that cryptochrome cannot repair UV-damaged DNA. The present investigation allowed us to conclude on the small but, apparently, critical differences between photolyase and cryptochrome. The performed analysis gives insight into important factors that govern the binding of UV-damaged DNA and reveal why cryptochrome cannot have this functionality.

  12. Erythrocyte 3H-ouabain binding and digitalis treatment in ethanol addicted patients

    International Nuclear Information System (INIS)

    Battaini, F.; Govoni, S.; Mauri, A.; Civelli, L.; Trabucchi, M.

    1987-01-01

    The binding of 3 H-ouabain to human erythrocytes was analyzed in a population of hospitalized male ethanol addicted patients under long term digitalis treatment. In the non-alcoholic patient group the long term digitalis treatment induced an increase in Bmax and Kd values; such modification was not observed in the alcoholic patients. Chronic alcohol intake itself induced an increase in 3 H-ouabain kinetic parameters. These observations confirm that ouabain binding to human erythrocytes is subject to pharmacological and toxicological regulation and that adaptive changes in peripheral tissues can be useful in predicting possible parallel modifications in other less accessible tissues. 22 references, 1 table

  13. Specificity of the Cyclic GMP-Binding Activity and of a Cyclic GMP-Dependent Cyclic GMP Phosphodiesterase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Walsum, Hans van; Meer, Rob C. van der; Bulgakov, Roman; Konijn, Theo M.

    1982-01-01

    The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H] cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at

  14. Displacement of specific serotonin and lysergic acid diethylamide binding by Ergalgin, a new antiserotonin drug

    International Nuclear Information System (INIS)

    Oelszner, W.

    1980-01-01

    [ 3 H]-serotonin and [ 3 H]-lysergic acid diethylamide (LSD) bind with a high affinity, Ksub(D) = 12 nM and 6 nM, respectively, to distinct receptors of rat caudate membranes in vitro. Displacement experiments with unlabeled serotonin and LSD support the hypothesis of serotonin receptors existing in an agonist and antagonist state. Methysergide and Ergalgin display quite similar potenties in displacing [ 3 H]-serontonin and [ 3 H]-LSD from their specific binding sites (Ksub(i) = 46.7 and 53.4 nM; 22.3 and 36.5 nM, respectively). Contrary to pharmacological findings these binding results are in favour of mixed agonist/antagonist properties of these compounds. (author)

  15. CHRONIC ALCOHOL NEUROADAPTATION AND STRESS CONTRIBUTE TO SUSCEPTIBILITY FOR ALCOHOL CRAVING AND RELAPSE

    Science.gov (United States)

    BREESE, GEORGE R.; SINHA, RAJITA; HEILIG, MARKUS

    2010-01-01

    Alcoholism is a chronic relapsing disorder. Major characteristics observed in alcoholics during an initial period of alcohol abstinence are altered physiological functions and a negative emotional state. Evidence suggests that a persistent, cumulative adaptation involving a kindling/allostasis-like process occurs during the course of repeated chronic alcohol exposures that is critical for the negative symptoms observed during alcohol withdrawal. Basic studies have provided evidence for specific neurotransmitters within identified brain sites being responsible for the negative emotion induced by the persistent cumulative adaptation following intermittent-alcohol exposures. After an extended period of abstinence, the cumulative alcohol adaptation increases susceptibility to stress- and alcohol cue-induced negative symptoms and alcohol seeking, both of which can facilitate excessive ingestion of alcohol. In the alcoholic, stressful imagery and alcohol cues alter physiological responses, enhance negative emotion, and induce craving. Brain fMRI imaging following stress and alcohol cues has documented neural changes in specific brain regions of alcoholics not observed in social drinkers. Such altered activity in brain of abstinent alcoholics to stress and alcohol cues is consistent with a continuing ethanol adaptation being responsible. Therapies in alcoholics found to block responses to stress and alcohol cues would presumably be potential treatments by which susceptibility for continued alcohol abuse can be reduced. By continuing to define the neurobiological basis of the sustained alcohol adaptation critical for the increased susceptibility of alcoholics to stress and alcohol cues that facilitate craving, a new era is expected to evolve in which the high rate of relapse in alcoholism is minimized. 250 PMID:20951730

  16. Alteration of gene expression by alcohol exposure at early neurulation.

    Science.gov (United States)

    Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

    2011-02-21

    We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube

  17. On binding specificity of (6–4) photolyase to a T(6–4)T DNA photoproduct

    DEFF Research Database (Denmark)

    Aalbæk Jepsen, Katrine; Solov'yov, Ilia

    2017-01-01

    this binding for a specific enzyme called (6–4) photolyase, which is capable of repairing certain UV-induced damage in DNA. Through molecular dynamics simulations we describe the binding between photolyase and the DNA and reveal that several charged amino acid residues in the enzyme, such as arginines...

  18. Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

    Science.gov (United States)

    Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

    2003-03-01

    Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

  19. Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells

    International Nuclear Information System (INIS)

    Tsujimoto, M.; Yip, Y.K.; Vilcek, J.

    1985-01-01

    Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125 I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125 I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 x 10 -10 M and 3.2 x 10 -10 M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37 0 C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125 I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF

  20. A randomized trial of female-specific cognitive behavior therapy for alcohol dependent women.

    Science.gov (United States)

    Epstein, Elizabeth E; McCrady, Barbara S; Hallgren, Kevin A; Cook, Sharon; Jensen, Noelle K; Hildebrandt, Thomas

    2018-02-01

    This study compared Female-Specific Cognitive Behavioral Therapy (FS-CBT) to evidence-based, gender-neutral CBT (GN-CBT; Epstein & McCrady, 2009) for women with alcohol use disorder (AUD). Women (N = 99) with AUD, mean age 48, were randomly assigned to 12 outpatient manual-guided sessions of FS-CBT (n = 44) or GN-CBT (n = 55). Women were assessed at baseline and 3, 9 and 15 months after baseline for drinking and for specific issues common among women with AUD. A FS-CBT protocol was developed that was discriminable on treatment integrity ratings from GN-CBT. No treatment condition differences were found in treatment engagement, changes in drinking, alcohol-related coping, abstinence self-efficacy, motivation to change, or constructs directly targeted in FS-CBT (sociotropy, autonomy, depression, anxiety). Women in both conditions were highly engaged and satisfied with treatment, and reported significant reductions in drinking and changes in desired directions for all other variables except social support for abstinence. In the year following treatment, women in the FS-CBT but not in the CBT condition reported an increase in percentage of abstainers in their social networks (0.69% per month, SE = 0.21, p = .002). The value and appeal of female-specific programming in AUD treatment has been established in the wider literature (Epstein & Menges, 2013), and the current study provides support for the use of the Female-Specific Cognitive Behavioral Therapy (FS-CBT) manual as an option that may yield outcomes similar to standard gender-neutral CBT for women with AUD. Future research should examine whether FS-CBT enhances treatment utilization for women. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

  1. Drinking game play among first-year college student drinkers: an event-specific analysis of the risk for alcohol use and problems.

    Science.gov (United States)

    Ray, Anne E; Stapleton, Jerod L; Turrisi, Rob; Mun, Eun-Young

    2014-09-01

    College students who play drinking games (DGs) more frequently report higher levels of alcohol use and experience more alcohol-related harm. However, the extent to which they are at risk for increased consumption and harm as a result of DG play on a given event after accounting for their typical DG participation, and typical and event drinking, is unclear. We examined whether first-year students consumed more alcohol and were more likely to experience consequences on drinking occasions when they played DGs. Participants (n = 336) completed up to six web-based surveys following weekend drinking events in their first semester. Alcohol use, DG play, and consequences were reported for the Friday and Saturday prior to each survey. Typical DG tendencies were controlled in all models. Typical and event alcohol use were controlled in models predicting risk for consequences. Participants consumed more alcohol on DG versus non-DG events. All students were more likely to experience blackout drinking consequences when they played DGs. Women were more likely to experience social-interpersonal consequences when they played DGs. DG play is an event-specific risk factor for increased alcohol use among first-year students, regardless of individual DG play tendencies. Further, event DG play signals increased risk for blackout drinking consequences for all students, and social-interpersonal consequences for women, aside from the amount of alcohol consumed on those occasions as well as typical drinking behaviors. Prevention efforts to reduce high-risk drinking may be strengthened by highlighting both event- and person-specific risks of DG play.

  2. /sup 125/I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of /sup 125/I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class AB diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more /sup 125/I-hEGF than did fetal membranes (P<0.0001). There was no significant differnce in /sup 125/I-hEGF binding to fetal membranes from the various pregnancy states (P<0.05). /sup 125/I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P<0.05). The binding to placentas from pregnancies complicated by White class AB diabetes or large for gestational age infants, on the other hand, was not significantly different from that to placentas from normal and appropriate for gestational age pregnancies. /sup 125/I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P<0.05). Placental and fetal membrane /sup 125/I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P<0.05). Placental but not fetal membrane /sup 125/I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone.

  3. Reactivity of solvent alcohol on degradation of CFC113

    International Nuclear Information System (INIS)

    Nakagawa, Seiko

    2003-01-01

    1,1,2-Trichloro-trifluoroethane (CFC113) was dissolved in alkaline 1-butanol, 2-butanol, iso-butyl alcohol, and phenyl ethyl alcohol and irradiated with 60 Co gamma rays after purged with pure nitrogen gas. In all these solvents, the concentration of CFC113 and hydroxide ion decreased and that of chloride ion increased with a dose observed in 2-propanol solution. The reaction efficiency increases in order of 1-butanol< iso-butyl alcohol< phenyl ethyl alcohol<2-butanol<2-propanol. The solvent effect will depend on the binding energy of the αC-H of the alcohol molecule and electron affinity and dipole moment of the ketones or aldehydes produced from the alcohols

  4. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

  5. Modulation of DNA binding by gene-specific transcription factors.

    Science.gov (United States)

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  6. The establishment and primary application of specific fatty acid binding protein radioimmunoassay

    International Nuclear Information System (INIS)

    Yan Guangtao; Zhang Kai; Xue Hui; Hao Xiuhua; Wang Luhuan

    2004-01-01

    A highly specific and highly sensitive radioimmunoassay method for fatty acid binding protein (FABP) in human serum was developed. The highly effective antibody againist FABP was obtained by immunizing rabbits with human recombined FABP. The FABP was labeled with 125 I by chloramines-T methods and purified by the Sephadex-G25 column. Te reaction between antigen and antibody was carried out by one step balance method and incubated in 4 degree for 24 hours, then binding and free antigen were separated by PR reagent. The detection range of this method is f rom 5 to 405 ng/mL; the lowest detection level is 9 ng/mL. CV's within batch and between batch were less than 6.4% and 8.5% respectively. The serum FABP concentration of healthy persons is 15.68 ± 2.91 ng/mL (n=45), that of ICU patients is 72.08 ± 32.64 ng/mL (n=46) and that of cardiopathy patients is 78.95 ± 24.83 ng/mL (n=73). It suggest that this method is stable, simple, specific and highly sensitive. It is suitable for testing FABP in human serum. (authors)

  7. Specificity of genetic and environmental risk factors for symptoms of cannabis, cocaine, alcohol, caffeine, and nicotine dependence.

    Science.gov (United States)

    Kendler, Kenneth S; Myers, John; Prescott, Carol A

    2007-11-01

    Although genetic risk factors have been found to contribute to dependence on both licit and illicit psychoactive substances, we know little of how these risk factors interrelate. To clarify the structure of genetic and environmental risk factors for symptoms of dependence on cannabis, cocaine, alcohol, caffeine, and nicotine in males and females. Lifetime history by structured clinical interview. General community. Four thousand eight hundred sixty-five members of male-male and female-female pairs from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. Main Outcome Measure Lifetime symptoms of abuse of and dependence on cannabis, cocaine, alcohol, caffeine, and nicotine. Controlling for greater symptom prevalence in males, genetic and environmental parameters could be equated across sexes. Two models explained the data well. The best-fit exploratory model contained 2 genetic factors and 1 individual environmental factor contributing to all substances. The first genetic factor loaded strongly on cocaine and cannabis dependence; the second, on alcohol and nicotine dependence. Nicotine and caffeine had high substance-specific genetic effects. A confirmatory model, which also fit well, contained 1 illicit drug genetic factor--loading only on cannabis and cocaine--and 1 licit drug genetic factor loading on alcohol, caffeine, and nicotine. However, these factors were highly intercorrelated (r = + 0.82). Large substance-specific genetic effects remained for nicotine and caffeine. The pattern of genetic and environmental risk factors for psychoactive substance dependence was similar in males and females. Genetic risk factors for dependence on common psychoactive substances cannot be explained by a single factor. Rather, 2 genetic factors-one predisposing largely to illicit drug dependence, the other primarily to licit drug dependence-are needed. Furthermore, a large proportion of the genetic influences on nicotine and particularly caffeine dependence

  8. In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

    Science.gov (United States)

    Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

    2013-11-15

    Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

  9. Relationship of binding specificity and structural property of the technetium-99m complexes for tumor hypoxia and tumor angiogenesis imaging

    International Nuclear Information System (INIS)

    Su, Z.F.

    2005-01-01

    The growth of tumor requires nutrition and oxygen. Tumor cells will become hypoxic when the supply of oxygen is insufficient. Hypoxic tumor cells will not only resist radiation therapy and chemotherapy, but also induce angiogenesis for oxygen supply and for metastasis. Therefore, detection of tumor hypoxia and tumor angiogenesis with high sensitive radio labeled imaging agents is important. Hypoxic tumor cells may display some molecules as tumor markers for the specific binding with radiopharmaceuticals. Radiopharmaceuticals, unlike the non-radioactive drugs, are trace compounds in a given dosage. Due to the extreme low concentration, the non-specific accumulation of the radiotracers by blood cells and proteins, tissues, and organs can be even more serious compared to the non-radioactive drugs. The non-specific accumulation of the radiotracers can make the ratios of tumor/tissue (in terms of i.d.%/g) falling to the range of 2∼7 [1-2]. Non-specific binding of radiopharmaceuticals is common, but detailed studies on it are poor documented. This presentation reports the study of the relationship of non-specific accumulation and the structural property of two type of 99m TC labeled compounds: (a) 99m Tc-(amine o xime) containing either 2-nitroimidazole (2-NI, as hypoxia tumor cells specific agents), or 4-nitro- imidazole (4-NI, as control), or aniline (as reference) groups; (b) 99m Tc-(arginine-glycine- aspartic acid, RGD, as tumor angiogenesis specific agents) and 99m Tc-(arginine-glycine- glutarmic acid, RGE, as control). The 99m Tc-(amine-oxime) complexes, in addition to the 2-NI, 4-NI, and aniline groups, contain methyl-, ethyl-, propyl-, iso-butyl-, t-butyl-, phenyl-, and Benzyl- groups as well to make the radiotracers differing in structure and in lipophilicity , while the lipophilicity of a radiotracer plays an important role in non-specific cellular accumulation and protein binding, The results demonstrated that (1) the complex containing 2-NI showed specific

  10. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  11. Impact of Maryland's 2011 alcohol sales tax increase on alcoholic beverage sales.

    Science.gov (United States)

    Esser, Marissa B; Waters, Hugh; Smart, Mieka; Jernigan, David H

    2016-07-01

    Increasing alcohol taxes has proven effective in reducing alcohol consumption, but the effects of alcohol sales taxes on sales of specific alcoholic beverages have received little research attention. Data on sales are generally less subject to reporting biases than self-reported patterns of alcohol consumption. We aimed to assess the effects of Maryland's July 1, 2011 three percentage point increase in the alcohol sales tax (6-9%) on beverage-specific and total alcohol sales. Using county-level data on Maryland's monthly alcohol sales in gallons for 2010-2012, by beverage type, multilevel mixed effects multiple linear regression models estimated the effects of the tax increase on alcohol sales. We controlled for seasonality, county characteristics, and national unemployment rates in the main analyses. In the 18 months after the tax increase, average per capita sales of spirits were 5.1% lower (p sales were 3.2% lower (p sales were 2.5% lower (p sales trends in the 18 months prior to the tax increase. Overall, the alcohol sales tax increase was associated with a 3.8% decline in total alcohol sold relative to what would have been expected based on sales in the prior 18 months (p increased alcohol sales taxes may be as effective as excise taxes in reducing alcohol consumption and related problems. Sales taxes also have the added advantages of rising with inflation and taxing the highest priced beverages most heavily.

  12. Rational steering of insulin binding specificity by intra-chain chemical crosslinking

    Science.gov (United States)

    Viková, Jitka; Collinsová, Michaela; Kletvíková, Emília; Buděšínský, Miloš; Kaplan, Vojtěch; Žáková, Lenka; Veverka, Václav; Hexnerová, Rozálie; Aviñó, Roberto J. Tarazona; Straková, Jana; Selicharová, Irena; Vaněk, Václav; Wright, Daniel W.; Watson, Christopher J.; Turkenburg, Johan P.; Brzozowski, Andrzej M.; Jiráček, Jiří

    2016-01-01

    Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

  13. Simultaneous localization of an hepatic binding protein specific for galactose and of galactose-containing receptors on rat hepatocytes.

    Science.gov (United States)

    Horisberger, M; VonLanthen, M

    1978-11-01

    The hepatic binding protein, specific for galactose-terminated glycoproteins (asialoglycoproteins) and the receptors for the Ricinus communis lectin, specific for galactose residues (RCA1), were simultaneously localized on isolated rat hepatocytes by the gold method. The marker for the binding protein was prepared from gold granules (5 nm in diam.) labeled with ceruloplasmin and desialylated. The marker specific for galactose-containing receptors consisted of granules (17 nm in diameter) labeled with RCA1. It was established that both markers did not interact. Hepatocytes (fresh or briefly fixed with glutaraldehyde) were successively incubated with the asialoceruloplasmin and the RCA1 marker. Examination of thin sections by electron microscopy indicated that the binding protein and the RCA1 receptors were often in the proximity of each other on the plasmamembrane. Using the same technique, wheat germ agglutinin (WGA) receptors were generally found on area of the plasmamembrane poorly marked by the RCA1 gold marker. The binding of asialoceruloplasmin gold markers was studied as a function of the size of the granules. It became insignificant when the size was above 17 nm. Previous results have shown that the binding of RCA1 is low when the marker reaches 50 nm in size while WGA markers up to 75 nm are well bound by hepatocytes. It is therefore hypothesized that the binding protein and RCA1 receptors are located between glycoprotein brushes of increasing spacing while part or all of the WGA receptors are located at the periphery of the brushes.

  14. The relationship between population-level exposure to alcohol advertising on television and brand-specific consumption among underage youth in the US.

    Science.gov (United States)

    Ross, Craig S; Maple, Emily; Siegel, Michael; DeJong, William; Naimi, Timothy S; Padon, Alisa A; Borzekowski, Dina L G; Jernigan, David H

    2015-05-01

    We investigated the population-level relationship between exposure to brand-specific advertising and brand-specific alcohol use among US youth. We conducted an internet survey of a national sample of 1031 youth, ages 13-20, who had consumed alcohol in the past 30 days. We ascertained all of the alcohol brands respondents consumed in the past 30 days, as well as which of 20 popular television shows they had viewed during that time period. Using a negative binomial regression model, we examined the relationship between aggregated brand-specific exposure to alcohol advertising on the 20 television shows [ad stock, measured in gross rating points (GRPs)] and youth brand-consumption prevalence, while controlling for the average price and overall market share of each brand. Brands with advertising exposure on the 20 television shows had a consumption prevalence about four times higher than brands not advertising on those shows. Brand-level advertising elasticity of demand varied by exposure level, with higher elasticity in the lower exposure range. The estimated advertising elasticity of 0.63 in the lower exposure range indicates that for each 1% increase in advertising exposure, a brand's youth consumption prevalence increases by 0.63%. At the population level, underage youths' exposure to brand-specific advertising was a significant predictor of the consumption prevalence of that brand, independent of each brand's price and overall market share. The non-linearity of the observed relationship suggests that youth advertising exposure may need to be lowered substantially in order to decrease consumption of the most heavily advertised brands. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  15. Lifetime total and beverage specific - alcohol intake and prostate cancer risk: a case-control study

    Directory of Open Access Journals (Sweden)

    Carruba Giuseppe

    2004-12-01

    Full Text Available Abstract Background We investigated lifetime alcohol consumption and prostate cancer risk in a case-control study conducted in Buffalo, NY (1998–2001. Methods The study included 88 men, aged 45 to 85 years with incident, histologically-confirmed prostate cancer and 272 controls. We conducted extensive in-person interviews regarding lifetime alcohol consumption and other epidemiologic data. Results Prostate cancer risk was not associated with lifetime intake of total and beverage specific ethanol. In addition we found no association with number of drinks per day (average drinks per day over the lifetime or drinks per drinking day (average drinks per day on drinking days only over the lifetime. However, we observed an inverse association with the total number of drinking years. Men in the lowest tertile of total drinking years had a two-fold prostate cancer risk than men in the highest tertile (OR 2.16, 95% CI 0.98–4.78, p for trend Conclusion Our results suggest that alcohol intake distribution across lifetime may play a more important role in prostate cancer etiology than total lifetime consumption.

  16. Current hypotheses on the mechanisms of alcoholism.

    Science.gov (United States)

    Vetreno, R P; Crews, F T

    2014-01-01

    Chronic use of alcohol results in progressive changes to brain and behavior that often lead to the development of alcohol dependence and alcoholism. Although the mechanisms underlying the development of alcoholism remain to be fully elucidated, diminished executive functioning due to hypoactive prefrontal cortex executive control and hyperactive limbic system anxiety and negative emotion might contribute mechanistically to the shift from experimental use to alcoholism and dependence. In the chapter that follows, behavioral deficits associated with cortical dysfunction and neurodegeneration will be related to the behavioral characteristics of alcoholism (e.g., diminished executive function, impulsivity, altered limbic modulation). We will provide evidence that alterations in cyclic AMP-responsive element binding protein (CREB: neurotrophic) and NF-κB (neuroimmune) signaling contribute to the development and persistence of alcoholism. In addition, genetic predispositions and an earlier age of drinking onset will be discussed as contributing factors to the development of alcohol dependence and alcoholism. Overall chronic ethanol-induced neuroimmune gene induction is proposed to alter limbic and frontal neuronal networks contributing to the development and persistence of alcoholism. © 2014 Elsevier B.V. All rights reserved.

  17. Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

    International Nuclear Information System (INIS)

    Gould, R.M.; Plapp, B.V.

    1990-01-01

    Molecular modeling of alcohol dehydrogenases suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degree C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD + and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver β enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD + complex limiting for turnover with ethanol

  18. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

  19. DNA-binding specificity and molecular functions of NAC transcription factors

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi Asschenfeldt; Lo Leggio, Leila

    2005-01-01

    The family of NAC (NAM/ATAF1,2/CUC2) transcription factors has been implicated in a wide range of plant processes, but knowledge on the DNA-binding properties of the family is limited. Using a reiterative selection procedure on random oligonucleotides, we have identified consensus binding sites....... Furthermore, NAC protein binding to the CaMV 35S promoter was shown to depend on sequences similar to the consensus of the selected oligonucleotides. Electrophoretic mobility shift assays demonstrated that NAC proteins bind DNA as homo- or heterodimers and that dimerization is necessary for stable DNA binding....... The ability of NAC proteins to dimerize and to bind DNAwas analysed by structure-based mutagenesis. This identified two salt bridge-forming residues essential for NAC protein dimerization. Alteration of basic residues in a loop region containing several highly conserved residues abolished DNA binding. Thus...

  20. A Multidimensional Model of Mothers’ Perceptions of Parent Alcohol Socialization and Adolescent Alcohol Misuse

    Science.gov (United States)

    Ennett, Susan T.; Jackson, Christine; Cole, Veronica T.; Haws, Susan; Foshee, Vangie A.; Reyes, Heathe Luz McNaughton; Burns, Alison Reimuller; Cox, Melissa J.; Cai, Li

    2015-01-01

    We assessed a multidimensional model of parent alcohol socialization in which key socialization factors were considered simultaneously to identify combinations of factors that increase or decrease risk for development of adolescent alcohol misuse. Of interest was the interplay between putative risk and protective factors, such as whether the typically detrimental effects on youth drinking of parenting practices tolerant of some adolescent alcohol use are mitigated by an effective overall approach to parenting and parental modeling of modest alcohol use. The sample included 1,530 adolescents and their mothers; adolescents’ mean age was 13.0 (SD = .99) at the initial assessment. Latent profile analysis was conducted of mothers’ reports of their attitude toward teen drinking, alcohol-specific parenting practices, parental alcohol use and problem use, and overall approach to parenting. The profiles were used to predict trajectories of adolescent alcohol misuse from early to middle adolescence. Four profiles were identified: two profiles reflected conservative alcohol-specific parenting practices and two reflected alcohol-tolerant practices, all in the context of other attributes. Alcohol misuse accelerated more rapidly from grade 6 through 10 in the two alcohol-tolerant compared with conservative profiles. Results suggest that maternal tolerance of some youth alcohol use, even in the presence of dimensions of an effective parenting style and low parental alcohol use and problem use, is not an effective strategy for reducing risky adolescent alcohol use. PMID:26415053

  1. Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro

    Directory of Open Access Journals (Sweden)

    Caixia Gao

    2017-12-01

    Full Text Available Swine leukocyte antigen (SLA class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs. Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m using splicing overlap extension polymerase chain reaction (SOE-PCR. The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV and four porcine reproductive and respiratory syndrome virus (PRRSV epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an

  2. Expanding the Substrate Specificity of Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual Site Mutation

    KAUST Repository

    Musa, Musa M.; Bsharat, Odey; Karume, Ibrahim; Vieille, Claire; Takahashi, Masateru; Hamdan, Samir

    2017-01-01

    Here, we report the asymmetric reduction of selected phenyl-ring-containing ketones by various single and dual site mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Further expanding the size of the substrate binding pocket in the mutant W110A/I86A not only allowed substrates of the single mutants W110A and I86A to be accommodated within the expanded active site, but also expanded the enzyme's substrate range to ketones bearing two sterically demanding groups (bulky-bulky ketones), which are not substrates for TeSADH single mutants. We also report the regio- and enantioselective reduction of diketones using W110A/I86A TeSADH and single TeSADH mutants. The double mutant exhibited dual stereopreference generating the Prelog products most of the time and the anti-Prelog products in a few cases.

  3. Expanding the Substrate Specificity of Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual Site Mutation

    KAUST Repository

    Musa, Musa M.

    2017-12-14

    Here, we report the asymmetric reduction of selected phenyl-ring-containing ketones by various single and dual site mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Further expanding the size of the substrate binding pocket in the mutant W110A/I86A not only allowed substrates of the single mutants W110A and I86A to be accommodated within the expanded active site, but also expanded the enzyme\\'s substrate range to ketones bearing two sterically demanding groups (bulky-bulky ketones), which are not substrates for TeSADH single mutants. We also report the regio- and enantioselective reduction of diketones using W110A/I86A TeSADH and single TeSADH mutants. The double mutant exhibited dual stereopreference generating the Prelog products most of the time and the anti-Prelog products in a few cases.

  4. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    Science.gov (United States)

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

    Science.gov (United States)

    Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

    1996-07-15

    Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

  6. Towards bilirubin imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) for the specific binding of α-bilirubin

    International Nuclear Information System (INIS)

    Syu, M.-J.; Deng, J.-H.; Nian, Y.-M.

    2004-01-01

    With α-bilirubin as a molecular template, polymerization of methacrylic acid (MAA) was carried out with the aid of the initiator 2,2-azobisisobutyronitrile (AIBN) and the cross-linking agent ethylene glycol dimethylacrylate (EGDMA). Bulk polymerization was successfully carried out so that poly(methacrylic acid-co-ethylene glycol dimethylacrylate) (poly(MAA-EGDMA)) imprinted with α-bilirubin was first developed. UV irradiation polymerization and heated polymerization methods were compared. Effect of different ratios of monomer to EGDMA during the polymerization was also discussed. Proper solvent for better desorption of α-bilirubin from the imprinted poly(MAA-EGDMA) was investigated. In addition, SEM photos were provided for observing the differences between the surfaces of the imprinted poly(MAA-EGDMA) before and after extraction. The corresponding binding results of α-bilirubin imprinted poly(MAA-EGDMA) and non-imprinted poly(MAA-EGDMA) both after extraction were compared. How the pH values during extraction stage affected the binding capacities of the imprinted polymer as well as non-imprinted polymer were also discussed. Similar study and comparison were made for different binding pH values. Different compounds of similar molecular weight were used to show the specific binding of the imprinted polymer for bilirubin. The results further confirmed the successful binding as well as specificity of the imprinted poly(MAA-EGDMA) for α-bilirubin

  7. Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

    Directory of Open Access Journals (Sweden)

    Xueyong Zhu

    2015-11-01

    Full Text Available Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

  8. Characterization of the binding specificity of Anguilla anguilla agglutinin (AAA) in comparison to Ulex europaeus agglutinin I (UEA-I).

    Science.gov (United States)

    Baldus, S E; Thiele, J; Park, Y O; Hanisch, F G; Bara, J; Fischer, R

    1996-08-01

    Using immunochemical and immunohistochemical methods, the binding site of Anguilla anguilla agglutinin (AAA) was characterized and compared with the related fucose-specific lectin from Ulex europaeus (UEA-I). In solid-phase enzyme-linked immunoassays, the two lectins recognized Fuc alpha 1-2Gal beta-HSA. AAA additionally cross-reacted with neoglycolipids bearing lacto-N-fucopentaose (LNFP) I [H type 1] and II [Le(a)] and lactodifucotetraose (LDFT) as glycan moieties. UEA-I, on the other hand, bound to a LDFT-derived neoglycolipid but not to the other neoglycolipids tested. Binding of AAA to gastric mucin was competitively neutralized by Le(a)-specific monoclonal antibodies. UEA-I binding, on the other hand, was reduced after co-incubation with H type 2- and Le(y)-specific monoclonal antibodies. According to our results, AAA reacts with fucosylated type 1 chain antigens, whereas UEA-I binds only to the alpha 1-2-fucosylated LDFT-derived neoglycolipid. In immunohistochemical studies, the reactivity of AAA and UEA-I in normal pyloric mucosa from individuals with known Lewis and secretor status was analysed. AAA showed a broad reaction in the superficial pyloric mucosa from secretors and non-secretors, but AAA reactivity was more pronounced in Le(a+b-) individuals. On the other hand, UEA-I stained the superficial pyloric mucosa only from secretor individuals. A staining of deep mucous glands by the lectins was found in all specimens. Both reacted with most human carcinomas of different origin. Slight differences in their binding pattern were observed and may be explained by the different fine-specificities of the lectins.

  9. Positive cooperativity of the specific binding between Hg2+ ion and T:T mismatched base pairs in duplex DNA

    International Nuclear Information System (INIS)

    Torigoe, Hidetaka; Miyakawa, Yukako; Ono, Akira; Kozasa, Tetsuo

    2012-01-01

    Highlights: ► Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio. ► The binding constant between Hg 2+ and the T:T mismatched base pair was 10 6 M −1 . ► The binding constant was larger than those for nonspecific metal–DNA interactions. ► The binding constant for the second Hg 2+ was larger than that for the first Hg 2+ . ► The positive cooperative binding was observed between Hg 2+ and multiple T:T. - Abstract: Metal-mediated base pairs by the interaction between metal ions and artificial bases in oligonucleotides have been developed for their potential applications in nanotechnology. We recently found that a natural T:T mismatched base pair bound with Hg 2+ ion to form a novel T–Hg–T base pair. Here, we examined the thermodynamic properties of the binding between Hg 2+ and each of the single and double T:T mismatched base pair duplex DNAs by isothermal titration calorimetry. Hg 2+ specifically bound with the T:T mismatched base pair at 1:1 molar ratio with 10 6 M −1 binding constant, which was significantly larger than those for nonspecific metal ion–DNA interactions. In the Hg 2+ –double T:T mismatched base pair interaction, the affinity for the second Hg 2+ binding was significantly larger than that for the first Hg 2+ binding. The positively cooperative binding may be favorable to align multiple Hg 2+ in duplex DNA for the application of the metal-mediated base pairs in nanotechnology.

  10. Measuring specific receptor binding of a PET radioligand in human brain without pharmacological blockade: The genomic plot.

    Science.gov (United States)

    Veronese, Mattia; Zanotti-Fregonara, Paolo; Rizzo, Gaia; Bertoldo, Alessandra; Innis, Robert B; Turkheimer, Federico E

    2016-04-15

    PET studies allow in vivo imaging of the density of brain receptor species. The PET signal, however, is the sum of the fraction of radioligand that is specifically bound to the target receptor and the non-displaceable fraction (i.e. the non-specifically bound radioligand plus the free ligand in tissue). Therefore, measuring the non-displaceable fraction, which is generally assumed to be constant across the brain, is a necessary step to obtain regional estimates of the specific fractions. The nondisplaceable binding can be directly measured if a reference region, i.e. a region devoid of any specific binding, is available. Many receptors are however widely expressed across the brain, and a true reference region is rarely available. In these cases, the nonspecific binding can be obtained after competitive pharmacological blockade, which is often contraindicated in humans. In this work we introduce the genomic plot for estimating the nondisplaceable fraction using baseline scans only. The genomic plot is a transformation of the Lassen graphical method in which the brain maps of mRNA transcripts of the target receptor obtained from the Allen brain atlas are used as a surrogate measure of the specific binding. Thus, the genomic plot allows the calculation of the specific and nondisplaceable components of radioligand uptake without the need of pharmacological blockade. We first assessed the statistical properties of the method with computer simulations. Then we sought ground-truth validation using human PET datasets of seven different neuroreceptor radioligands, where nonspecific fractions were either obtained separately using drug displacement or available from a true reference region. The population nondisplaceable fractions estimated by the genomic plot were very close to those measured by actual human blocking studies (mean relative difference between 2% and 7%). However, these estimates were valid only when mRNA expressions were predictive of protein levels (i

  11. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao

    2003-01-01

    terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also...... recognizes both main families of repeat sequences in S. solfataricus. The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match...... with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus-specific protein induces an opening of the structure...

  12. Beverage- and Brand-Specific Binge Alcohol Consumption among Underage Youth in the U.S.

    Science.gov (United States)

    Naimi, Timothy S; Siegel, Michael; DeJong, William; O'Doherty, Catherine; Jernigan, David

    2015-09-01

    Binge drinking is a common and risky pattern of alcohol consumption among youth; beverage and brand-specific consumption during binge drinking is poorly understood. The objective was to characterize beverage- and brand-specific consumption associated with binge drinking among underage youth in the U.S. An internet panel was used to obtain a sample of 1,032 underage youth aged 13-20, who drank alcohol in the past 30 days. For each brand consumed, youth reported drinking quantity and frequency, and whether they engaged in binge drinking with that brand (≥5 drinks for males, ≥4 for females). Each youth reporting binge drinking with a brand constituted a binge drinking report. Overall, 50.9% of youth binge drank with ≥1 brand, and 36.5% of youth who consumed any particular brand reported binge drinking with it. Spirits accounted for 43.8% of binge drinking reports. Twenty-five brands accounted for 46.2% of binge drinking reports. Many of these brands were disproportionately associated with binge drinking relative to their youth market share. Binge drinking among youth is most commonly involves spirits, and binge drinking is concentrated within a relatively small number of brands. Understanding factors underlying beverage and brand preference among binge drinking youth could assist prevention efforts.

  13. High non-specific binding of the {beta}{sub 1}-selective radioligand 2-{sup 125}I-ICI-H

    Energy Technology Data Exchange (ETDEWEB)

    Riemann, B. [Muenster Univ. (Germany). Department of Nuclear Medicine; Law, M.P. [Muenster Univ. (Germany). Department of Nuclear Medicine; Hammersmith Hospital, London (United Kingdom). MRC Clinical Sciences Centre; Kopka, K. [Muenster Univ. (DE). Department of Nuclear Medicine] [and others

    2003-08-01

    Aim: As results of cardiac biopsies suggest, myocardial {beta}{sub 1}-adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac {beta}{sub 2}-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess {beta}-adrenoceptors non-invasively. Among the novel racemic analogues of the established {beta}{sub 1}-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to {beta}{sub 1}-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative subtype selective {beta}{sub 1}-adrenergic radioligand in cardiac imaging. Methods: Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac {beta}-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-{sup 125}I-ICI-H from the silylated precursor 2-SiMe{sub 3}-ICI-H was established. The specific activity was 80 GBq/{mu}mol, the radiochemical yield ranged from 70 to 80%. Results: The unlabelled compound 2-I-ICI-H showed high {beta}{sub 1}-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac {beta}-adrenoceptors. The radiolabelled counterpart 2-{sup 125}I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac {beta}{sub 1}-adrenoceptors in vivo. Conclusion: Because of its high non-specific binding 2-{sup 125}I-ICI-H is no suitable radiotracer for imaging in vivo. (orig.)

  14. Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Ahring, Philip K; Olsen, Jeppe A

    2015-01-01

    Neuronal α4β2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(β2)(2) receptor subpopulation was discovered. In particular, three......-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the α4-α4 interface. Confirmed by mutational studies, our structures suggest a unique ligand-specific role of residue H142 on the α4 subunit. In the cocrystal...... that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting...

  15. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  16. Specific binding of 125I-salmon calcitonin to rat brain

    International Nuclear Information System (INIS)

    Nakamuta, Hiromichi; Furukawa, Shinichi; Koida, Masao; Yajima, Haruaki; Orlowski, R.C.

    1981-01-01

    Rat brain particulate fraction was found to contain binding sites for 125 I-Salmon Calcitonin-I ( 125 I-SCT). Maximum binding occurred in the physiological pH range of 7.25 - 7.5. The binding reaction proceeded in a temperature-dependent manner. Binding sites were broadly distributed among the various rat brain regions and considerable regional differences existed in the affinity and density as detected by Scatchard analysis. The highest affinity was recorded in the case of the hypothalamus and the lowest in the case of the cerebellum. The KD (nM) and Bmax (pmole/mg protein) estimated for the binding to four regions were as follows: hypothalamus: 1.4 and 0.19, midbrain, hippocampus plus striatum: 1.5 and 0.08, pon plus medulla oblongata: 3.0 and 0.15 and cerebellum: 8.3 and 0.20. Using a particulate fraction of rat brain void of cerebellum and cortices, a binding assay for calcitonins was developed. Binding of 125 I-SCT was inhibited by unlabeled salmon, [Asu sup(1,7)]-eel and porcine calcitonins in a dose-dependent manner and the IC50s were 2.0, 8.0 and 30 nM, respectively. The IC50s were comparable to those estimated using a kidney particulate fraction. Human calcitonin, β-endorphin and substance P were weak inhibitors of the binding. Other peptides, drugs and putative neurotransmitters tested (totally 23 substances) failed to inhibit the binding at concentrations of 1.0 μM. The physiological significance of brain binding sites for calcitonin, with the possibility that the brain may possess endogenous ligands for these sites are discussed. (author)

  17. Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

    Science.gov (United States)

    Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

    1993-01-01

    SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

  18. Binge Alcohol Exposure Transiently Changes the Endocannabinoid System: A Potential Target to Prevent Alcohol-Induced Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Daniel J. Liput

    2017-11-01

    Full Text Available Excessive alcohol consumption leads to neurodegeneration, which contributes to cognitive decline that is associated with alcohol use disorders (AUDs. The endocannabinoid system has been implicated in the development of AUDs, but little is known about how the neurotoxic effects of alcohol impact the endocannabinoid system. Therefore, the current study investigated the effects of neurotoxic, binge-like alcohol exposure on components of the endocannabinoid system and related N-acylethanolamines (NAEs, and then evaluated the efficacy of fatty acid amide hydrolase (FAAH inhibition on attenuating alcohol-induced neurodegeneration. Male rats were administered alcohol according to a binge model, which resulted in a transient decrease in [3H]-CP-55,940 binding in the entorhinal cortex and hippocampus following two days, but not four days, of treatment. Furthermore, binge alcohol treatment did not change the tissue content of the three NAEs quantified, including the endocannabinoid and anandamide. In a separate study, the FAAH inhibitor, URB597 was administered to rats during alcohol treatment and neuroprotection was assessed by FluoroJade B (FJB staining. The administration of URB597 during binge treatment did not significantly reduce FJB+ cells in the entorhinal cortex or hippocampus, however, a follow up “target engagement” study found that NAE augmentation by URB597 was impaired in alcohol intoxicated rats. Thus, potential alcohol induced alterations in URB597 pharmacodynamics may have contributed to the lack of neuroprotection by FAAH inhibition.

  19. Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins

    Institute of Scientific and Technical Information of China (English)

    HSU Heng; Robert L. CHURCH

    2004-01-01

    During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a "CA" repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preliminarily identify trans factors which bind to specific cis elements of the LIM2 promoter and/or regulate transcription of LIM2. We conclude that developmental regulation and coordination of the MP 19 gene in ocular lens fiber cells is controlled by the presence of specific cis elements that bind regulatory trans factors that affect LIM2 gene expression. DNA methylation is one mechanism of controlling LIM2 gene expression during lens development.

  20. A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

    Science.gov (United States)

    Miller, A

    1977-01-01

    The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

  1. A multidimensional model of mothers' perceptions of parent alcohol socialization and adolescent alcohol misuse.

    Science.gov (United States)

    Ennett, Susan T; Jackson, Christine; Cole, Veronica T; Haws, Susan; Foshee, Vangie A; Reyes, Heathe Luz McNaughton; Burns, Alison Reimuller; Cox, Melissa J; Cai, Li

    2016-02-01

    We assessed a multidimensional model of parent alcohol socialization in which key socialization factors were considered simultaneously to identify combinations of factors that increase or decrease risk for development of adolescent alcohol misuse. Of interest was the interplay between putative risk and protective factors, such as whether the typically detrimental effects on youth drinking of parenting practices tolerant of some adolescent alcohol use are mitigated by an effective overall approach to parenting and parental modeling of modest alcohol use. The sample included 1,530 adolescents and their mothers; adolescents' mean age was 13.0 (SD = .99) at the initial assessment. Latent profile analysis was conducted of mothers' reports of their attitude toward teen drinking, alcohol-specific parenting practices, parental alcohol use and problem use, and overall approach to parenting. The profiles were used to predict trajectories of adolescent alcohol misuse from early to middle adolescence. Four profiles were identified: 2 profiles reflected conservative alcohol-specific parenting practices and 2 reflected alcohol-tolerant practices, all in the context of other attributes. Alcohol misuse accelerated more rapidly from Grade 6 through 10 in the 2 alcohol-tolerant compared with conservative profiles. Results suggest that maternal tolerance of some youth alcohol use, even in the presence of dimensions of an effective parenting style and low parental alcohol use and problem use, is not an effective strategy for reducing risky adolescent alcohol use. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  2. Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

    Directory of Open Access Journals (Sweden)

    Kareem Mohideen-Abdul

    2017-06-01

    Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half

  3. Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

    International Nuclear Information System (INIS)

    Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini

    2011-01-01

    Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

  4. Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

    International Nuclear Information System (INIS)

    Adámik, Matej; Bažantová, Pavla; Navrátilová, Lucie; Polášková, Alena; Pečinka, Petr; Holaňová, Lucie; Tichý, Vlastimil; Brázdová, Marie

    2015-01-01

    Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed

  5. Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

    Energy Technology Data Exchange (ETDEWEB)

    Adámik, Matej [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Bažantová, Pavla [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Navrátilová, Lucie; Polášková, Alena [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Pečinka, Petr [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Holaňová, Lucie [Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic); Tichý, Vlastimil [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Brázdová, Marie, E-mail: maruska@ibp.cz [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic)

    2015-01-02

    Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.

  6. Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

    Science.gov (United States)

    Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

    2012-10-15

    Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. The Relationship Between Population-Level Exposure to Alcohol Advertising on Television and Brand-Specific Consumption Among Underage Youth in the US

    Science.gov (United States)

    Ross, Craig S.; Maple, Emily; Siegel, Michael; DeJong, William; Naimi, Timothy S.; Padon, Alisa A.; Borzekowski, Dina L.G.; Jernigan, David H.

    2015-01-01

    Aims: We investigated the population-level relationship between exposure to brand-specific advertising and brand-specific alcohol use among US youth. Methods: We conducted an internet survey of a national sample of 1031 youth, ages 13–20, who had consumed alcohol in the past 30 days. We ascertained all of the alcohol brands respondents consumed in the past 30 days, as well as which of 20 popular television shows they had viewed during that time period. Using a negative binomial regression model, we examined the relationship between aggregated brand-specific exposure to alcohol advertising on the 20 television shows [ad stock, measured in gross rating points (GRPs)] and youth brand-consumption prevalence, while controlling for the average price and overall market share of each brand. Results: Brands with advertising exposure on the 20 television shows had a consumption prevalence about four times higher than brands not advertising on those shows. Brand-level advertising elasticity of demand varied by exposure level, with higher elasticity in the lower exposure range. The estimated advertising elasticity of 0.63 in the lower exposure range indicates that for each 1% increase in advertising exposure, a brand's youth consumption prevalence increases by 0.63%. Conclusions: At the population level, underage youths' exposure to brand-specific advertising was a significant predictor of the consumption prevalence of that brand, independent of each brand's price and overall market share. The non-linearity of the observed relationship suggests that youth advertising exposure may need to be lowered substantially in order to decrease consumption of the most heavily advertised brands. PMID:25754127

  8. Fractures and alcohol abuse - patient opinion of alcohol intervention

    DEFF Research Database (Denmark)

    Pedersen, Bolette; Alva-Jørgensen, Peter; Raffing, Rie

    2011-01-01

    PURPOSE: To clarify patient opinions about alcohol intervention in relation to surgery before investigating the effect in a Scandinavian multi-centre randomized trial. MATERIAL AND METHODS: A qualitative study. Thirteen consecutive alcohol patients with fractures participated after informed consent....... They were interviewed during their hospital stay. The number of participants was based on the criteria of data-saturation. The analysis followed the applied qualitative framework model aimed at evaluation of specific participant needs within a larger overall project. RESULTS: All patients regarded alcohol...

  9. Are there gender differences in locus of control specific to alcohol dependence?

    Science.gov (United States)

    McPherson, Andrew; Martin, Colin R

    2017-01-01

    To investigate gender differences in locus of control in an alcohol-dependent population. Locus of control helps to explain behaviour in terms of internal (the individual is responsible) or external (outside forces, such as significant other people or chance, are responsible) elements. Past research on gender differences in locus of control in relation to alcohol dependence has shown mixed results. There is a need then to examine gender and locus of control in relation to alcohol dependence to ascertain the veracity of any locus of control differences as a function of gender. The Multidimensional Health Locus of Control form-C was administered to clients from alcohol dependence treatment centres in the West of Scotland. Independent t-tests were carried out to assess gender differences in alcohol dependence severity and internal/external aspects of locus of control. One hundred and eighty-eight (53% females) participants were recruited from a variety of alcohol dependence treatment centres. The majority of participants (72%) came from Alcoholics Anonymous groups. Women revealed a greater internal locus of control compared with men. Women also had a greater 'significant others' locus of control score than men. Men were more reliant on 'chance' and 'doctors' than women. All these trends were not, however, statistically significant. Gender differences in relation to locus of control and alcohol dependence from past studies are ambiguous. This study also found no clear statistically significant differences in locus of control orientation as a function of gender. This article helps nurses to contextualise health behaviours as a result of internal or external forces. It also helps nursing staff to better understand alcohol dependence treatment in relation to self-efficacy and control. Moreover, it highlights an important concept in health education theory. © 2016 John Wiley & Sons Ltd.

  10. Corticotropin-Releasing Factor (CRF) Neurocircuitry and Neuropharmacology in Alcohol Drinking.

    Science.gov (United States)

    Schreiber, Allyson L; Gilpin, Nicholas W

    2018-01-28

    Alcohol use is pervasive in the United States. In the transition from nonhazardous drinking to hazardous drinking and alcohol use disorder, neuroadaptations occur within brain reward and brain stress systems. One brain signaling system that has received much attention in animal models of excessive alcohol drinking and alcohol dependence is corticotropin-releasing factor (CRF). The CRF system is composed of CRF, the urocortins, CRF-binding protein, and two receptors - CRF type 1 and CRF type 2. This review summarizes how acute, binge, and chronic alcohol dysregulates CRF signaling in hypothalamic and extra-hypothalamic brain regions and how this dysregulation may contribute to changes in alcohol reinforcement, excessive alcohol consumption, symptoms of negative affect during withdrawal, and alcohol relapse. In addition, it summarizes clinical work examining CRF type 1 receptor antagonists in humans and discusses why the brain CRF system is still relevant in alcohol research.

  11. Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

    International Nuclear Information System (INIS)

    Poli, M.A.

    1985-01-01

    The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C 42 aldehyde function of T34 to the alcohol function of T17 using NaB 3 H 4 yielded 3 H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4 0 CC, 22 0 C, and 37 0 C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles 3 H-T17 per mg of synaptosomal protein at 4 0 C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace 3 H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that 3 H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed

  12. Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

    Energy Technology Data Exchange (ETDEWEB)

    Poli, M.A.

    1985-01-01

    The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C/sub 42/ aldehyde function of T34 to the alcohol function of T17 using NaB/sup 3/H/sub 4/ yielded /sup 3/H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4/sup 0/CC, 22/sup 0/C, and 37/sup 0/C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles /sup 3/H-T17 per mg of synaptosomal protein at 4/sup 0/C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace /sup 3/H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that /sup 3/H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed.

  13. Strain-Specific Virolysis Patterns of Human Noroviruses in Response to Alcohols.

    Science.gov (United States)

    Park, Geun Woo; Collins, Nikail; Barclay, Leslie; Hu, Liya; Prasad, B V Venkataram; Lopman, Benjamin A; Vinjé, Jan

    2016-01-01

    Alcohol-based hand sanitizers are widely used to disinfect hands to prevent the spread of pathogens including noroviruses. Alcohols inactivate norovirus by destruction of the viral capsid, resulting in the leakage of viral RNA (virolysis). Since conflicting results have been reported on the susceptibility of human noroviruses against alcohols, we exposed a panel of 30 human norovirus strains (14 GI and 16 GII strains) to different concentrations (50%, 70%, 90%) of ethanol and isopropanol and tested the viral RNA titer by RT-qPCR. Viral RNA titers of 10 (71.4%), 14 (100%), 3 (21.4%) and 7 (50%) of the 14 GI strains were reduced by > 1 log10 RNA copies/ml after exposure to 70% and 90% ethanol, and 70% and 90% isopropanol, respectively. RNA titers of 6 of the 7 non-GII 4 strains remained unaffected after alcohol exposure. Compared to GII strains, GI strains were more susceptible to ethanol than to isopropanol. At 90%, both alcohols reduced RNA titers of 8 of the 9 GII.4 strains by ≥ 1 log10 RNA copies/ml. After exposure to 70% ethanol, RNA titers of GII.4 Den Haag and Sydney strains decreased by ≥ 1.9 log10, whereas RNA reductions for GII.4 New Orleans strains were alcohol susceptibility patterns between different norovirus genotypes vary widely and that virolysis data for a single strain or genotype are not representative for all noroviruses.

  14. Alcohol Alert: Genetics of Alcoholism

    Science.gov (United States)

    ... daily rhythm for various functions (e.g., body temperature or blood pressure) that is controlled by certain “ ... A special section delves more deeply into specific classes of genes and their relationship to alcoholism. The ...

  15. 98 Specific IGE and IGG Binding to Allergoids of Phleum pratense

    OpenAIRE

    Cases, Barbara; Fernandez-Caldas, Enrique; Tudela, Jose Ignacio; Fernandez, Eva Abel; Sanchez-Garcia, Silvia; Ibañez, M. Dolores; Escudero, Carmelo; Casanovas, Miguel

    2012-01-01

    Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods Th...

  16. 27 CFR 21.116 - Methyl alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Methyl alcohol. 21.116 Section 21.116 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

  17. 27 CFR 21.113 - Isopropyl alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Isopropyl alcohol. 21.113 Section 21.113 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

  18. Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as revealed by X-ray crystallography, NMR and MD simulations.

    Directory of Open Access Journals (Sweden)

    Xuelu Huan

    Full Text Available The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD, which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity

  19. Brain-specific inactivation of the Crhr1 gene inhibits post-dependent and stress-induced alcohol intake, but does not affect relapse-like drinking

    DEFF Research Database (Denmark)

    Molander, Anna; Vengeliene, Valentina; Heilig, Markus

    2012-01-01

    , a conditional brain-specific Crhr1-knockout (Crhr1(NestinCre)) and a global knockout mouse line were studied for basal alcohol drinking, stress-induced alcohol consumption, deprivation-induced intake, and escalated alcohol consumption in the post-dependent state. In a second set of experiments, we tested CRHR1...... not affect relapse-like drinking after a deprivation period in rats. We conclude that CRH/CRHR1 extra-HPA and HPA signaling may have opposing effects on stress-related alcohol consumption. CRHR1 does not have a role in basal alcohol intake or relapse-like drinking situations with a low stress load.......Corticotropin-releasing hormone (CRH) and its receptor, CRH receptor-1 (CRHR1), have a key role in alcoholism. Especially, post-dependent and stress-induced alcohol intake involve CRH/CRHR1 signaling within extra-hypothalamic structures, but a contribution of the hypothalamic-pituitary-adrenal (HPA...

  20. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  1. Alcohol Expectancies Mediate and Moderate the Associations between Big Five Personality Traits and Adolescent Alcohol Consumption and Alcohol-Related Problems.

    Science.gov (United States)

    Ibáñez, Manuel I; Camacho, Laura; Mezquita, Laura; Villa, Helena; Moya-Higueras, Jorge; Ortet, Generós

    2015-01-01

    Personality and expectancies are relevant psychological factors for the development of adolescent alcohol use and misuse. The present study examined their direct, mediated and moderated effects on different drinking behaviors in adolescence. Personality domains of the five-factor model, positive and negative alcohol expectancies (AEs), alcohol use during the week and the weekend, and alcohol-related problems were assessed in a sample of 361 adolescents. Different personality dimensions were directly associated with specific alcohol outcomes: Extraversion, low Conscientiousness and low Openness were associated with weekend alcohol use; low Agreeableness was related to weekday use; whereas low Agreeableness, low Conscientiousness and Extraversion were associated with alcohol-related problems. In addition, positive AEs mediated the relationship between Extraversion and alcohol use, whereas both positive and negative expectancies mediated the association between Neuroticism and alcohol consumption and alcohol-related problems. Finally, both types of expectancies interacted with Extraversion to predict alcohol problems. Our results highlight the importance of examining the complex interplay of comprehensive personality models and AEs to gain a better understanding of the development of different alcohol use and misuse patterns in adolescence.

  2. Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ford, K.A.; LaBarbera, A.R.

    1988-11-01

    The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase in receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.

  3. Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin.

    Science.gov (United States)

    Kumar, Santosh; Bose, Debojit; Suryawanshi, Hemant; Sabharwal, Harshana; Mapa, Koyeli; Maiti, Souvik

    2011-01-01

    Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT(m) = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K(a) = 16.2±0.6×10(7) M(-1) where enthalpic change ΔH = -13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = -2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K(a) = 3.1±0.4×10(7) M(-1)) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.

  4. Defining Alcohol-Specific Rules Among Parents of Older Adolescents: Moving Beyond No Tolerance.

    Science.gov (United States)

    Bourdeau, Beth; Miller, Brenda; Vanya, Magdalena; Duke, Michael; Ames, Genevieve

    2012-01-01

    Parental beliefs and rules regarding their teen's use of alcohol influence teen decisions regarding alcohol use. However, measurement of parental rules regarding adolescent alcohol use has not been thoroughly studied. This study used qualitative interviews with 174 parents of older teens from 100 families. From open-ended questions, themes emerged that describe explicit rules tied to circumscribed use, no tolerance, and "call me." There was some inconsistency in explicit rules with and between parents. Responses also generated themes relating to implicit rules such as expectations and preferences. Parents described their methods of communicating their position via conversational methods, role modeling their own behavior, teaching socially appropriate use of alcohol by offering their teen alcohol, and monitoring their teens' social activities. Findings indicate that alcohol rules are not adequately captured by current assessment measures.

  5. ALCOHOL AND HEART RHYTHM DISORDERS

    Directory of Open Access Journals (Sweden)

    A. O. Yusupova

    2015-01-01

    Full Text Available Alcohol abuse and particularly extension of alcohol consumption in alcohol diseas increases the risk of cardiac arrhythmias development and aggravates existing arrhythmias. Patients do not always receive the necessary specific treatment due to lack of detection of the ethanol genesis of these arrhythmias. Management of patients with alcohol abuse and alcohol dependence, including its cardiac complications among other cardiac arrhythmias should use both antiarrhythmic and anti-alcohol drugs and antidepressants. Such issues as diagnosis and management of patients with alcohol-induced cardiac arrhythmias are presented.

  6. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting.

    Science.gov (United States)

    Wu, Shwu-Yuan; Lee, A-Young; Lai, Hsien-Tsung; Zhang, Hong; Chiang, Cheng-Ming

    2013-03-07

    Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Prolactin receptors in liver, kidney, and gill of the tilapia (Oreochromis mossambicus): Characterization and effect of salinity on specific binding of iodinated ovine prolactin

    International Nuclear Information System (INIS)

    Dauder, S.; Young, G.; Hass, L.; Bern, H.A.

    1990-01-01

    Specific binding of 125 I-ovine prolactin (oPRL) to microsomal fractions from gill, kidney, and liver of adult tilapia was determined. Specific binding varied among tissues, the highest values being displayed by kidney membranes. In the liver, the binding of oPRL was not strongly displaced by tilapia prolactins (tPRL177 and tPRL188), although tPRL177 was six times more potent than tPRL188. On the other hand, in kidney and gill membranes, the two tPRLs were equipotent. Tilapia PRLs showed low potency in competing for oPRL-binding sites when pregnant rat liver membranes were utilized. Tilapia growth hormone (tGH) and human growth hormone (hGH) displaced 125 I-oPRL from liver as well as did tPRL177 but were not recognized well by renal or branchial receptors. Two 125 I-oPRL-binding sites were detected in every tissue tested. These binding sites are subject to physiological regulation since adaptation to seawater resulted in a significant decrease in specific binding

  8. Heavy Episodic Drinking and Alcohol-Related Consequences: Sex-Specific Differences in Parental Influences among Ninth-Grade Students

    Science.gov (United States)

    Doumas, Diana M.; Hausheer, Robin; Esp, Susan

    2015-01-01

    Parents impact adolescent substance abuse, but sex-specific influences are not well-understood. This study examined parental influences on adolescent drinking behavior in a sample of ninth-grade students (N = 473). Hierarchical regression analyses indicated parental monitoring, disapproval of teen alcohol use, and quality of parent-teen general…

  9. An investigation of strategies used in alcohol brand marketing and alcohol-related health promotion on Facebook.

    Science.gov (United States)

    Lim, Megan Sc; Hare, James D; Carrotte, Elise R; Dietze, Paul M

    2016-01-01

    Alcohol brands are incorporating social networking sites (SNS) into their marketing programmes. SNS are also being used to reduce alcohol consumption and harms by health promotion organisations. Marketing via SNS can attempt to influence consumers using a range of strategies from traditional marketing, social media, and behaviour change theory. This study systematically quantifies marketing strategies used by alcohol brands and health promoters on Facebook. We identified the 10 most popular alcohol brands and health promotion organisations in Australia on Facebook and extracted all posts from April 2014. A framework was developed, listing 33 SNS marketing strategies. The frequency of use of each strategy in posts was counted for all profiles. The median number of fans of alcohol brands was 189,290 compared with 7562 for health promotion pages. A total of 210 Facebook posts were analysed. Popular marketing strategies included visual attraction, connecting with other organisations, and links to culture and events. Time-specific and day-specific posts and tweets were used more regularly by alcohol brands than health promotion agencies. Alcohol brands remain substantially more popular than health promotion organisations, and this difference is likely driven by offline factors rather than specific use of marketing strategies. However, health promotion organisations can learn from the strategies used by popular brands, particularly in the use of time and day-specific content.

  10. Interaction of alcohols with the calcite surface

    DEFF Research Database (Denmark)

    Bovet, Nicolas Emile; Yang, Mingjun; Javadi, Meshkat Sadat

    2015-01-01

    . Controlling their growth requires complex polysaccharides. Polysaccharide activity depends on the functionality of OH groups, so to simplify the system in order to get closer to a molecular level understanding, we investigated the interaction of OH from a suite of alcohols with clean, freshly cleaved calcite...... surfaces. X-ray photoelectron spectroscopy (XPS) provided binding energies and revealed the extent of surface coverage. Molecular dynamics (MD) simulations supplemented with information about molecule ordering, orientation and packing density. The results show that all alcohols studied bond...... with the calcite surface through the OH group, with their carbon chains sticking away in a standing-up orientation. Alcohol molecules are closely packed and form a well-ordered monolayer on the surface....

  11. Organ specific acute toxicity of the carcinogen trans-4-acetylaminostilbene is not correlated with macromolecular binding.

    Science.gov (United States)

    Pfeifer, A; Neumann, H G

    1986-09-01

    trans-4-Acetylaminostilbene (trans-AAS) is acutely toxic in rats and lesions are produced specifically in the glandular stomach. Toxicity is slightly increased by pretreating the animals with phenobarbital (PB) and is completely prevented by pretreatment with methylcholanthrene (MC). The prostaglandin inhibitors, indomethacin and acetyl salicylic acid, do not reduce toxicity. The high efficiency of MC suggested that toxicity is caused by reactive metabolites. trans-[3H]-AAS was administered orally to untreated and to PB- or MC-pretreated female Wistar rats and target doses in different tissues were measured by means of covalent binding to proteins, RNA and DNA. Macromolecular binding in the target tissue of poisoned animals was significantly lower than in liver and kidney and comparable to other non-target tissues. Pretreatment with MC lowered macromolecular binding in all extrahepatic tissues but not in liver. These findings are not in line with tissue specific metabolic activation. The only unique property of the target tissue, glandular stomach, that we observed was a particular affinity for the systemically available parent compound. In the early phase of poisoning, tissue concentrations were exceedingly high and the stomach function was impaired.

  12. Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

    Science.gov (United States)

    Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

    2009-08-17

    Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

  13. Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

    Science.gov (United States)

    Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

    1989-01-01

    Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

  14. Specific binding of prostaglandin E2 to membrane preparations from human skin: receptor modulation by UVB-irradiation and chemical agents

    International Nuclear Information System (INIS)

    Lord, J.T.; Ziboh, V.A.

    1979-01-01

    Human skin membranes bind prostaglandin E2 (PGE2) with high affinity and specificity. This binding is inhibited by trypsin or heat treatment suggesting that PGE2 receptors have protein components. Exposure of the membranes to ultraviolet irradiation (UVB) resulted in the loss of the membrane binding capacity for PGE2. This UVB-inhibitory effect could be prevented by a known protein sulfhydryl-oxidizing agent and a known lipid anti-oxidant

  15. Specific binding-adsorbent assay method and test means

    International Nuclear Information System (INIS)

    1981-01-01

    A description is given of an improved specific binding assay method and test means employing a nonspecific adsorbent for the substance to be determined, particularly hepatitis B surface (HBsub(s)) antigen, in its free state or additionally in the form of its immune complex. The invention is illustrated by 1) the radioimmunoadsorbent assay for HBsub(s) antigen, 2) the radioimmunoadsorbent assay for HBsub(s) antigen in the form of immune complex with antibody, 3) a study of adsorption characteristics of various anion exchange materials for HBsub(s) antigen, 4) the use of hydrophobic adsorbents in a radioimmunoadsorbent assay for HBsub(s) antigen and 5) the radioimmunoadsorbent assay for antibody to HBsub(s) antigen. The advantages of the present method for detecting HBsub(s) antigen compared to previous methods include the manufacturing advantages of eliminating the need for insolubilised anti-HBsub(s) and the advantages of a single incubation step, fewer manipulations, storability of adsorbent materials, increased sensitivity and versatility of detecting HBsub(s) antigen in the form of its immune complex if desired. (U.K.)

  16. Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

    Science.gov (United States)

    Nallapareddy, Sreedhar R; Weinstock, George M; Murray, Barbara E

    2003-03-01

    A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the

  17. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

    Science.gov (United States)

    Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2015-08-01

    Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

  18. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Support Vector Machine (SVM) is a state-of-the-art classifica- tion technique. Using canonical binding model, the C2H2 zinc finger protein–DNA interaction interface is modelled by the pairwise amino acid–base interactions. Using a classification framework, known examples of non-binding ZF–DNA pairs.

  19. Effects of a Variety of Food Extracts and Juices on the Specific Binding Ability of Norovirus GII.4 P Particles

    Science.gov (United States)

    LI, DAN; BAERT, LEEN; XIA, MING; ZHONG, WEIMING; JIANG, XI; UYTTENDAELE, MIEKE

    2014-01-01

    The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks. PMID:22980024

  20. Effect of solvent alcohol on degradation of chlorinated hydrocarbons by γ-irradiation

    International Nuclear Information System (INIS)

    Nakagawa, Seiko

    2002-01-01

    1,1,2-Trichloro-trifluoroethane (CFC113) was dissolved in alkaline 1-butanol, 2-butanol, iso-butyl alcohol, and phenyl ethyl alcohol and irradiated with 60 Co gamma rays after being purged with pure nitrogen gas. In all these solvents, the concentration of CFC113 and hydroxide ion decreased and that of chloride ion increased with a dose observed in 2-propanol solution. The reaction efficiency increases in the following order: 1-butanol < iso-butyl alcohol < phenyl ethyl alcohol < 2-butanol < 2-propanol. The solvent effect will depend on the binding energy of the αC-H of the alcohol molecule and electron affinity and dipole moment of the ketones or aldehydes produced from the alcohols. (author)

  1. Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces.

    Science.gov (United States)

    Samra, H K; Ganguly, N K; Mahajan, R C

    1991-06-01

    The effects of human milk, containing specific secretory IgA, on the adherence of Giardia lamblia trophozoites in the presence and in the absence of intestinal mucus in vitro were studied. It was found that the trophozoites treated with breast milk, containing specific secretory IgA to G. lamblia, showed a significant decrease (p less than 0.01) in adherence to nylon fibre columns and glass surfaces than did trophozoites treated with milk containing no SIgA antibodies. The adherence to glass surfaces was significantly more (p less than 0.01) in the presence of intestinal mucus than when the mucus was absent. Milk that did not contain specific secretory SIgA to G. lamblia did not decrease the adherence to glass surfaces either in the presence or in the absence of mucus. The fluorescence study revealed the binding of specific secretory IgA on the trophozoite surface. The results suggest that binding of SIgA antibodies in milk to G. lamblia trophozoites inhibits parasite adherence, thus protecting against this infection in breast-fed babies.

  2. An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

    International Nuclear Information System (INIS)

    Rosenke, Kyle; Samuel, Melanie A.; McDowell, Eric T.; Toerne, Melissa A.; Fortunato, Elizabeth A.

    2006-01-01

    The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection

  3. Increased serum cortisol binding in chronic active hepatitis

    International Nuclear Information System (INIS)

    Orbach, O.; Schussler, G.C.

    1989-01-01

    A high serum cortisol concentration, apparently due to increased cortisol-binding globulin (CBG), was found in a patient (index case) with chronic active hepatitis (CAH). We therefore performed further studies to determine whether increased cortisol binding is generally associated with CAH. Serum samples were obtained from 15 hospitalized patients with long-term liver function test elevations but no evidence of cirrhosis, 15 normal subjects without a history of hepatitis, four healthy pregnant women, and 10 alcoholic patients with stigmata of cirrhosis. Serum cortisol binding was measured by an adaptation of a previously described charcoal uptake method. Thyroxine-binding globulin (TBG) and sex hormone-binding globulin were determined by radioimmunoassays. Charcoal uptake of 125I cortisol from sera of normal subjects and additional patients with CAH revealed that increased serum cortisol binding by a saturable site, presumably CBG, was associated with CAH. Cortisol binding was significantly correlated with immunoassayable TBG, suggesting that in CAH, similar mechanisms may be responsible for increasing the serum concentrations of CBG and TBG

  4. Binding of 3H-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    International Nuclear Information System (INIS)

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.

    1985-01-01

    Binding of 3 H-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method

  5. CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

    Science.gov (United States)

    Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

    2014-11-19

    Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired

  6. Associations of prodynorphin sequence variation with alcohol dependence and related traits are phenotype-specific and sex-dependent.

    Science.gov (United States)

    Winham, Stacey J; Preuss, Ulrich W; Geske, Jennifer R; Zill, Peter; Heit, John A; Bakalkin, Georgy; Biernacka, Joanna M; Karpyak, Victor M

    2015-10-27

    We previously demonstrated that prodynorphin (PDYN) haplotypes and single nucleotide polymorphism (SNP) rs2281285 are associated with alcohol dependence and the propensity to drink in negative emotional states, and recent studies suggest that PDYN gene effects on substance dependence risk may be sex-related. We examined sex-dependent associations of PDYN variation with alcohol dependence and related phenotypes, including negative craving, time until relapse after treatment and the length of sobriety episodes before seeking treatment, in discovery and validation cohorts of European ancestry. We found a significant haplotype-by-sex interaction (p  =  0.03), suggesting association with alcohol dependence in males (p = 1E-4) but not females. The rs2281285 G allele increased risk for alcohol dependence in males in the discovery cohort (OR = 1.49, p = 0.002), with a similar trend in the validation cohort (OR = 1.35, p = 0.086). However, rs2281285 showed a trend towards association with increased negative craving in females in both the discovery (beta = 10.16, p = 0.045) and validation samples (OR = 7.11, p = 0.066). In the discovery cohort, rs2281285 was associated with time until relapse after treatment in females (HR = 1.72, p = 0.037); in the validation cohort, it was associated with increased length of sobriety episodes before treatment in males (beta = 13.49, p = 0.001). Our findings suggest that sex-dependent effects of PDYN variants in alcohol dependence are phenotype-specific.

  7. The kinetics of specific [3H]flunitrazepam ([3H]FNZ) binding in the brain of the epaulette shark (hemiscyllium ocellatum)

    International Nuclear Information System (INIS)

    Wise, G.; Renshaw, G.M.C.; Dodd, P.R.

    1998-01-01

    Full text: We have previously established that the epaulette shark is tolerant to hypoxia and that the resulting brain hypometabolism appears to be correlated with increased levels of GABA. It is of interest to determine whether there is a change in GABA A receptor number and/or binding characteristics in response to hypoxia. The focus of this initial study is to determine the kinetics of [ 3 H]FNZ binding to the benzodiazapine binding site on the GABA A receptor in the brain, so that the effect of hypoxia on GABA A receptors can be determined. Adult epaulette sharks were anaesthetised with 80mg/L of MS222 and the brain was dissected and rapidly frozen. Membranes were prepared at 4 deg C by homogenising the dissected tissue in 0.32M sucrose and centrifuging the homogenate for 10 min at 6 000g. The supernatant was layered onto an aliquat of 0.8M sucrose then centrifuged for 30 min at 40 000g. After washing the membranes, the binding characteristics of [ 3 H]FNZ were examined using in vitro centrifugation assays. This method revealed that [ 3 H]FNZ bound specifically to low-affinity binding sites in an elasmobranch brain. This finding is in contrast with previous reports of little to no specific binding of [ 3 H]FNZ in elasmobranchs, which precluded an estimation of binding parameters. Copyright (1998) Australian Neuroscience Society

  8. 27 CFR 21.101 - tert-Butyl alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false tert-Butyl alcohol. 21.101 Section 21.101 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

  9. 27 CFR 21.100 - n-Butyl alcohol.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false n-Butyl alcohol. 21.100 Section 21.100 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specifications for Denaturants § 21...

  10. Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

    Directory of Open Access Journals (Sweden)

    Michelle P Christie

    Full Text Available The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

  11. Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

    Science.gov (United States)

    Mouw, M; Pintel, D J

    1998-11-10

    GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

  12. Linearized method: A new approach for kinetic analysis of central dopamine D2 receptor specific binding

    International Nuclear Information System (INIS)

    Watabe, Hiroshi; Hatazawa, Jun; Ishiwata, Kiichi; Ido, Tatsuo; Itoh, Masatoshi; Iwata, Ren; Nakamura, Takashi; Takahashi, Toshihiro; Hatano, Kentaro

    1995-01-01

    The authors proposed a new method (Linearized method) to analyze neuroleptic ligand-receptor specific binding in a human brain using positron emission tomography (PET). They derived the linear equation to solve four rate constants, k 3 , k 4 , k 5 , k 6 from PET data. This method does not demand radioactivity curve in plasma as an input function to brain, and can do fast calculations in order to determine rate constants. They also tested Nonlinearized method including nonlinear equations which is conventional analysis using plasma radioactivity corrected for ligand metabolites as an input function. The authors applied these methods to evaluate dopamine D 2 receptor specific binding of [ 11 C] YM-09151-2. The value of B max /K d = k 3 k 4 obtained by Linearized method was 5.72 ± 3.1 which was consistent with the value of 5.78 ± 3.4 obtained by Nonlinearized method

  13. Macromolecular weight specificity in covalent binding of bromobenzene

    International Nuclear Information System (INIS)

    Sun, J.D.; Dent, J.G.

    1984-01-01

    Bromobenzene is a hepatotoxicant that causes centrilobular necrosis. Pretreatment of animals with 3-methylcholanthrene decreases and phenobarbital pretreatment enhances the hepatotoxic action of this compound. We have investigated the macromolecular weight specificity of the covalent interactions of bromobenzene with liver macromolecules following incubation of [ 14 C]bromobenzene in isolated hepatocytes. Hepatocytes were prepared from Fischer-344 rats treated for 3 days with 3-methylcholanthrene, phenobarbital, or normal saline. After a 1-hr incubation, total covalent binding, as measured by sodium dodecyl sulfate-equilibrium dialysis, was twofold less in hepatocytes from 3-methylcholanthrene-treated rats and sixfold greater in hepatocytes from phenobarbital-treated rats, as compared to hepatocytes from control animals. Analysis of the arylated macromolecules by electrophoresis on 15% sodium dodecyl sulfate-polyacrylamide disc gels indicated that in the first 1 to 3 min of incubation substantial amounts of covalently bound radiolabel were associated with macromolecules of between 20,000 and 40,000. The amount of radioactivity associated with these macromolecules rapidly diminished in hepatocytes from control and 3-methylcholanthrene-treated animals. In hepatocytes from phenobarbital-treated animals, the amount of radioactivity associated with macromolecules, 20,000, increased throughout the incubation. The amount of radiolabel associated with macromolecules, 20,000, increased in all incubations. When nontoxic doses of phenylmethylsulfonyl fluoride, a specific inhibitor of serine proteases, were added to control hepatocytes incubated with [ 14 C]-bromobenzene, the decrease in radioactivity associated with larger (greater than 20,000) macromolecules was inhibited and a corresponding lack of increase in radioactivity associated with smaller macromolecules was observed

  14. Peer deviance, alcohol expectancies, and adolescent alcohol use: explaining shared and nonshared environmental effects using an adoptive sibling pair design.

    Science.gov (United States)

    Samek, Diana R; Keyes, Margaret A; Iacono, William G; McGue, Matt

    2013-07-01

    Previous research suggests adolescent alcohol use is largely influenced by environmental factors, yet little is known about the specific nature of this influence. We hypothesized that peer deviance and alcohol expectancies would be sources of environmental influence because both have been consistently and strongly correlated with adolescent alcohol use. The sample included 206 genetically related and 407 genetically unrelated sibling pairs assessed in mid-to-late adolescence. The heritability of adolescent alcohol use (e.g., frequency, quantity last 12 months) was minimal and not significantly different from zero. The associations among peer deviance, alcohol expectancies, and alcohol use were primarily due to shared environmental factors. Of special note, alcohol expectancies also significantly explained nonshared environmental influence on alcohol use. This study is one of few that have identified specific environmental variants of adolescent alcohol use while controlling for genetic influence.

  15. Pathogen-Specific Binding Soluble Down Syndrome Cell Adhesion Molecule (Dscam Regulates Phagocytosis via Membrane-Bound Dscam in Crab

    Directory of Open Access Journals (Sweden)

    Xue-Jie Li

    2018-04-01

    Full Text Available The Down syndrome cell adhesion molecule (Dscam gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1–Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1–Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.

  16. Multiple POU-binding motifs, recognized by tissue-specific nuclear factors, are important for Dll1 gene expression in neural stem cells

    International Nuclear Information System (INIS)

    Nakayama, Kohzo; Nagase, Kazuko; Tokutake, Yuriko; Koh, Chang-Sung; Hiratochi, Masahiro; Ohkawara, Takeshi; Nakayama, Noriko

    2004-01-01

    We cloned the 5'-flanking region of the mouse homolog of the Delta gene (Dll1) and demonstrated that the sequence between nucleotide position -514 and -484 in the 5'-flanking region of Dll1 played a critical role in the regulation of its tissue-specific expression in neural stem cells (NSCs). Further, we showed that multiple POU-binding motifs, located within this short sequence of 30 bp, were essential for transcriptional activation of Dll1 and also that multiple tissue-specific nuclear factors recognized these POU-binding motifs in various combinations through differentiation of NSCs. Thus, POU-binding factors may play an important role in Dll1 expression in developing NSCs

  17. Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael

    2015-01-01

    with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped...

  18. Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA.

    Science.gov (United States)

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta G

    2016-02-29

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

    Science.gov (United States)

    Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

    2014-12-01

    Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

  20. Does last week's alcohol intake affect semen quality or reproductive hormones?

    DEFF Research Database (Denmark)

    Hansen, M L; Thulstrup, A M; Bonde, J P

    2012-01-01

    The association between last 5 days of alcohol intake, semen quality and reproductive hormones was estimated in this cross-sectional study among 347 men. Conventional semen characteristics, DNA fragmentation index and reproductive hormones (testosterone, estradiol, sex hormone-binding globulin...... (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B) were determined. There was a tendency towards lower semen characteristics at higher intake of alcohol past 5 days, albeit with no statistically significant dose-response association. The ratio between free estradiol...... and free testosterone was higher at higher alcohol intake during the 5 days preceding semen sampling. In conclusion, alcohol intake was associated with impairment of most semen characteristics but without a coherent dose-response pattern. The study indicates an association between recent alcohol intake...

  1. State Responses to Alcohol Use and Pregnancy: Findings From the Alcohol Policy Information System (APIS)

    OpenAIRE

    DRABBLE, LAURIE; THOMAS, SUE; O’CONNOR, LISA; ROBERTS, SARAH CM

    2014-01-01

    This article describes U.S. state policies related to alcohol use during pregnancy, using data from the National Institute on Alcohol Abuse and Alcoholism (NIAAA) Alcohol Policy Information System (APIS). Specifically, this study examines trends in policies enacted by states over time and types of policies enacted across states in the U.S., with a focus on whether laws were supportive or punitive toward women. Findings revealed substantial variability in characteristics of policies (19 primar...

  2. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  3. MATERNAL ALCOHOL CONSUMPTION PRODUCING FETAL ALCOHOL SPECTRUM DISORDERS (FASD): QUANTITY, FREQUENCY, AND TIMING OF DRINKING

    Science.gov (United States)

    May, Philip A.; Blankenship, Jason; Marais, Anna-Susan; Gossage, J. Phillip; Kalberg, Wendy O.; Joubert, Belinda; Cloete, Marise; Barnard, Ronel; De Vries, Marlene; Hasken, Julie; Robinson, Luther K.; Adnams, Colleen M.; Buckley, David; Manning, Melanie; Parry, Charles; Hoyme, H. Eugene; Tabachnick, Barbara; Seedat, Soraya

    2013-01-01

    Background Concise, accurate measures of maternal prenatal alcohol use are needed to better understand fetal alcohol spectrum disorders (FASD). Methods Measures of drinking by mothers of children with specific FASD diagnoses and mothers of randomly-selected controls are compared and also correlated with physical and cognitive/behavioral outcomes. Results Measures of maternal alcohol use can differentiate maternal drinking associated with FASD from that of controls and some from mothers of alcohol-exposed normals. Six variables that combine quantity and frequency concepts distinguish mothers of FASD children from normal controls. Alcohol use variables, when applied to each trimester and three months prior to pregnancy, provide insight on critical timing of exposure as well. Measures of drinking, especially bingeing, correlate significantly with increased child dysmorphology and negative cognitive/behavioral outcomes in children, especially low non-verbal IQ, poor attention, and behavioral problems. Logistic regression links (palcohol consumption both within and between diagnostic groupings of mothers bearing children diagnosed within the FASD continuum. Drinking measures are empirically identified and correlated with specific child outcomes. Alcohol use, especially heavy use, should be avoided throughout pregnancy. PMID:23932841

  4. Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity.

    Science.gov (United States)

    Zhang, Peilan; Li, Kunhua; Yang, Guang; Xia, Changqing; Polston, Jane E; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-Jun; Bruner, Steven D; Ding, Yousong

    2017-08-22

    Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.

  5. Variation in gastric alcohol dehydrogenase and the risk of alcohol dependence

    Directory of Open Access Journals (Sweden)

    Paulina Całka

    2017-03-01

    Full Text Available Alcohol dependence is both a medical and socioeconomic problem. The disease is multifactorial, i.e. its development is attributable to gene-gene and gene-environment interactions. Multi-centre studies investigating the genetic background of alcoholism stress the role of genes encoding enzymes of the ethanol decomposition pathway in the human body, particularly alcohol dehydrogenase (ADH, in the development of alcohol dependence. Among five classes of alcohol dehydrogenases, class I and IV isoenzymes have been found to be associated with alcohol dependence. Class IV is of particular interest due to its occurrence in the upper gastrointestinal tract, mainly in the stomach. No activity of the enzyme has been demonstrated in the liver. Single nucleotide polymorphism (SNP of the gene encoding ADH class IV (ADH7 affects its ethanol-oxidizing activity in the gastric lumen, thereby influencing the first-pass metabolism (FPM of the substance. The findings published by various research centres have demonstrated that specific SNP changes in the ADH7 gene are of different significance for the risk of alcohol dependence according to the population studied.

  6. College Men and Alcohol Use: Positive Alcohol Expectancies as a Mediator Between Distinct Masculine Norms and Alcohol Use.

    Science.gov (United States)

    Iwamoto, Derek Kenji; Corbin, William; Lejuez, Carl; MacPherson, Laura

    2014-01-01

    College men are more likely to engage in health-compromising behaviors including risky drinking behavior, and experience more alcohol-related problems, including violence and arrest, as compared to women. The study of masculine norms or societal expectations, defined as beliefs and values about what it means to be a man, is one promising area of investigation that may help explain within-group differences and differential rates of alcohol use among men. Using the gender social learning model, we investigated the role of positive alcohol expectancies as an underlying mediator between masculine norms and alcohol use among college men. Data from 804 college adult men ( Mean age = 20.43) were collected through a web-based assessment. Participants completed a self-report measure of binge drinking, frequency of drinking, quantity of drinks, conformity to masculine norms, and positive alcohol expectancies measures. Structural equation modeling was used to examine relations between masculine norms, alcohol expectancies and alcohol use. The masculine norms of "Playboy" and Risk-Taking were positively related to heavy alcohol use, while Emotional Control and Heterosexual Presentation were both negatively associated with alcohol use, after controlling for fraternity Greek status and positive expectancies. Playboy and Winning norms were positively associated with positive expectancies while Power Over Women was inversely related to positive expectancies which, in turn, were associated with heavier alcohol use. This study was a novel exploration into the multiple pathways and mediators through which positive alcohol expectancies may help explain and provide specificity to the masculinity and alcohol use relationship among college men.

  7. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

    KAUST Repository

    Li, Lixin; Atef, Ahmed; Piatek, Agnieszka Anna; Ali, Zahir; Piatek, Marek J.; Aouida, Mustapha; Sharakuu, Altanbadralt; Mahjoub, Ali; Wang, Guangchao; Khan, Mohammad Suhail; Fedoroff, Nina V.; Zhu, Jiankang; Mahfouz, Magdy M.

    2013-01-01

    , including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA

  8. Acute effect of intravenously applied alcohol in the human striatal and extrastriatal D2 /D3 dopamine system.

    Science.gov (United States)

    Pfeifer, Philippe; Tüscher, Oliver; Buchholz, Hans Georg; Gründer, Gerhard; Vernaleken, Ingo; Paulzen, Michael; Zimmermann, Ulrich S; Maus, Stephan; Lieb, Klaus; Eggermann, Thomas; Fehr, Christoph; Schreckenberger, Mathias

    2017-09-01

    Investigations on the acute effects of alcohol in the human mesolimbic dopamine D 2 /D 3 receptor system have yielded conflicting results. With respect to the effects of alcohol on extrastriatal D 2 /D 3 dopamine receptors no investigations have been reported yet. Therefore we applied PET imaging using the postsynaptic dopamine D 2 /D 3 receptor ligand [ 18 F]fallypride addressing the question, whether intravenously applied alcohol stimulates the extrastriatal and striatal dopamine system. We measured subjective effects of alcohol and made correlation analyses with the striatal and extrastriatal D 2 /D 3 binding potential. Twenty-four healthy male μ-opioid receptor (OPRM1)118G allele carriers underwent a standardized intravenous and placebo alcohol administration. The subjective effects of alcohol were measured with a visual analogue scale. For the evaluation of the dopamine response we calculated the binding potential (BP ND ) by using the simplified reference tissue model (SRTM). In addition, we calculated distribution volumes (target and reference regions) in 10 subjects for which metabolite corrected arterial samples were available. In the alcohol condition no significant dopamine response in terms of a reduction of BP ND was observed in striatal and extrastriatal brain regions. We found a positive correlation for 'liking' alcohol and the BP ND in extrastriatal brain regions (Inferior frontal cortex (IFC) (r = 0.533, p = 0.007), orbitofrontal cortex (OFC) (r = 0.416, p = 0.043) and prefrontal cortex (PFC) (r = 0.625, p = 0.001)). The acute alcohol effects on the D 2 /D 3 dopamine receptor binding potential of the striatal and extrastriatal system in our experiment were insignificant. A positive correlation of the subjective effect of 'liking' alcohol with cortical D 2 /D 3 receptors may hint at an addiction relevant trait. © 2016 Society for the Study of Addiction.

  9. Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

    Science.gov (United States)

    Wu, A M; Song, S C; Sugii, S; Herp, A

    1997-01-01

    Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E

  10. Sasquatch: predicting the impact of regulatory SNPs on transcription factor binding from cell- and tissue-specific DNase footprints.

    Science.gov (United States)

    Schwessinger, Ron; Suciu, Maria C; McGowan, Simon J; Telenius, Jelena; Taylor, Stephen; Higgs, Doug R; Hughes, Jim R

    2017-10-01

    In the era of genome-wide association studies (GWAS) and personalized medicine, predicting the impact of single nucleotide polymorphisms (SNPs) in regulatory elements is an important goal. Current approaches to determine the potential of regulatory SNPs depend on inadequate knowledge of cell-specific DNA binding motifs. Here, we present Sasquatch, a new computational approach that uses DNase footprint data to estimate and visualize the effects of noncoding variants on transcription factor binding. Sasquatch performs a comprehensive k -mer-based analysis of DNase footprints to determine any k -mer's potential for protein binding in a specific cell type and how this may be changed by sequence variants. Therefore, Sasquatch uses an unbiased approach, independent of known transcription factor binding sites and motifs. Sasquatch only requires a single DNase-seq data set per cell type, from any genotype, and produces consistent predictions from data generated by different experimental procedures and at different sequence depths. Here we demonstrate the effectiveness of Sasquatch using previously validated functional SNPs and benchmark its performance against existing approaches. Sasquatch is available as a versatile webtool incorporating publicly available data, including the human ENCODE collection. Thus, Sasquatch provides a powerful tool and repository for prioritizing likely regulatory SNPs in the noncoding genome. © 2017 Schwessinger et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Predicting women's alcohol risk-taking while abroad.

    Science.gov (United States)

    Smith, Gabie; Klein, Sarah

    2010-05-01

    Numerous studies have examined risk factors that are associated with heavy alcohol use; however, much of this research has not addressed factors that specifically relate to women's alcohol use. The current study has extended the previous literature on women's alcohol-use behavior by examining factors associated with risky drinking in young women traveling abroad (n = 55). Using a pretest-posttest design, we examined the influence of disinhibition sensation-seeking and endorsement of social enhancement alcohol expectancies in relation to participation in risky alcohol use while abroad for three weeks. Analyses confirmed that disinhibition sensation-seeking and social enhancement alcohol expectancies were associated with participation in risky alcohol-use behaviors while abroad (controlling for alcohol-use at the pretest). Analysis of qualitative data reinforced the importance of social facilitation in women's alcohol risk-taking. Participants' qualitative data also emphasized characteristics of situational disinhibition relating to travel as well as culturally-specific motivations for alcohol-use behaviors. Further research examining women's personal need for disinhibition and the role of situational disinhibition in motivating alcohol risk-taking is warranted. In addition, the current findings suggest that interventions focusing on the connections between alcohol use and enhancement of social relationships and the potential isolating effects of non-use are necessary.

  12. Effects of prenatal alcohol and cigarette exposure on offspring substance use in multiplex, alcohol-dependent families.

    Science.gov (United States)

    O'Brien, Jessica W; Hill, Shirley Y

    2014-12-01

    Prenatal exposures to alcohol, cigarettes, and other drugs of abuse are associated with numerous adverse consequences for affected offspring, including increased risk for substance use and abuse. However, maternal substance use during pregnancy appears to occur more often in those with a family history of alcohol dependence. Utilizing a sample that is enriched for familial alcohol dependence and includes controls selected for virtual absence of familial alcohol dependence could provide important information on the relative contribution of familial risk and prenatal exposures to offspring substance use. A sample of multigenerational families specifically ascertained to be at either high or low risk for developing alcohol dependence (AD) provided biological offspring for a longitudinal prospective study. High-risk families were selected based on the presence of 2 alcohol-dependent sisters. Low-risk families were selected on the basis of minimal first and second-degree relatives with AD. High-risk (HR = 99) and Low-risk offspring (LR = 110) were assessed annually during childhood and biennially in young adulthood regarding their alcohol, drug, and cigarette use. At the first childhood visit, mothers were interviewed concerning their prenatal use of substances. High-risk mothers were more likely to use alcohol, cigarettes, and other drugs during pregnancy than low-risk control mothers, and to consume these substances in greater quantities. Across the sample, prenatal exposure to alcohol was associated with increased risk for both offspring cigarette use and substance use disorders (SUD), and prenatal cigarette exposure was associated with increased risk for offspring cigarette use. Controlling for risk status by examining patterns within the HR sample, prenatal cigarette exposure remained a specific predictor of offspring cigarette use, and prenatal alcohol exposure was specifically associated with increased risk for offspring SUD. Women with a family history of

  13. 24 CFR 5.860 - When am I specifically authorized to evict alcohol abusers?

    Science.gov (United States)

    2010-04-01

    ... Federally Assisted Housing-Denying Admission and Terminating Tenancy for Criminal Activity or Alcohol Abuse... of alcohol threatens the health, safety, or right to peaceful enjoyment of the premises by other...

  14. Defining Alcohol-Specific Rules Among Parents of Older Adolescents: Moving Beyond No Tolerance

    OpenAIRE

    Bourdeau, Beth; Miller, Brenda; Vanya, Magdalena; Duke, Michael; Ames, Genevieve

    2012-01-01

    Parental beliefs and rules regarding their teen’s use of alcohol influence teen decisions regarding alcohol use. However, measurement of parental rules regarding adolescent alcohol use has not been thoroughly studied. This study used qualitative interviews with 174 parents of older teens from 100 families. From open-ended questions, themes emerged that describe explicit rules tied to circumscribed use, no tolerance, and “call me.” There was some inconsistency in explicit rules with and betwee...

  15. Genes, Environments, and Sex Differences in Alcohol Research.

    Science.gov (United States)

    Salvatore, Jessica E; Cho, Seung Bin; Dick, Danielle M

    2017-07-01

    The study of sex differences has been identified as one way to enhance scientific reproducibility, and the National Institutes of Health (NIH) have implemented a new policy to encourage the explicit examination of sex differences. Our goal here is to address sex differences in behavioral genetic research on alcohol outcomes. We review sex differences for alcohol outcomes and whether the source and magnitude of genetic influences on alcohol consumption and alcohol use disorder (AUD) are the same across sexes; describe common research designs for studying sex-specific gene-by-environment interaction (G × E) effects; and discuss the role of statistical power and theory when testing sex-specific genetic effects. There are robust sex differences for many alcohol outcomes. The weight of evidence suggests that the source and magnitude of genetic influences on alcohol consumption and AUD are the same across sexes. Whether there are sex-specific G × E effects has received less attention to date. The new NIH policy necessitates a systematic approach for studying sex-specific genetic effects in alcohol research. Researchers are encouraged to report power for tests of these effects and to use theory to develop testable hypotheses, especially for studies of G × E.

  16. Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

    Science.gov (United States)

    Couñago, Rafael M; Knapp, Karen M; Nakatani, Yoshio; Fleming, Stephen B; Corbett, Michael; Wise, Lyn M; Mercer, Andrew A; Krause, Kurt L

    2015-07-07

    The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a β-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP β-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP β-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity

    Science.gov (United States)

    Zhang, Peilan; Yang, Guang; Xia, Changqing; Polston, Jane E.; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-jun; Bruner, Steven D.

    2017-01-01

    Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan–GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus. Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1–4(Fucα1–3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment. PMID:28784797

  18. Molecular basis of alcoholism.

    Science.gov (United States)

    Most, Dana; Ferguson, Laura; Harris, R Adron

    2014-01-01

    Acute alcohol intoxication causes cellular changes in the brain that last for hours, while chronic alcohol use induces widespread neuroadaptations in the nervous system that can last a lifetime. Chronic alcohol use and the progression into dependence involve the remodeling of synapses caused by changes in gene expression produced by alcohol. The progression of alcohol use, abuse, and dependence can be divided into stages, which include intoxication, withdrawal, and craving. Each stage is associated with specific changes in gene expression, cellular function, brain circuits, and ultimately behavior. What are the molecular mechanisms underlying the transition from recreational use (acute) to dependence (chronic)? What cellular adaptations result in drug memory retention, leading to the persistence of addictive behaviors, even after prolonged drug abstinence? Research into the neurobiology of alcoholism aims to answer these questions. This chapter will describe the molecular adaptations caused by alcohol use and dependence, and will outline key neurochemical participants in alcoholism at the molecular level, which are also potential targets for therapy. © 2014 Elsevier B.V. All rights reserved.

  19. Carbon 14 and tritium radioactivity of alcohols

    International Nuclear Information System (INIS)

    Guerain, J.; Tourliere, S.

    1975-01-01

    The method of measuring carbon 14 radioactivity of alcohols has been perfected in order to establish the correct determination of synthetic alcohol added to fermentation alcohol. The specific carbon and tritium activity of alcohol of different origins have been determined for 1973 and 1974. The Suess effect and nuclear fall-out are observed [fr

  20. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Community College Student Alcohol Use: Developing Context-Specific Evidence and Prevention Approaches

    Science.gov (United States)

    Wall, Andrew F.; BaileyShea, Chelsea; McIntosh, Scott

    2012-01-01

    The objective of this study was to examine the prevalence of heavy alcohol use, related harm, and implications for prevention among community college students. We used data from 7,965 students at 19 community colleges who responded to the Core Alcohol and Other Drug Survey. This secondary analysis of the survey data found heavy consumption among…

  2. Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

    DEFF Research Database (Denmark)

    Kold, David; Dauter, Zbigniew; Laustsen, Anne K

    2014-01-01

    The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds...... of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but...

  3. Prospective Study of Alcohol Consumption Quantity and Frequency and Cancer-Specific Mortality in the US Population

    Science.gov (United States)

    Breslow, Rosalind A.; Chen, Chiung M.; Graubard, Barry I.; Mukamal, Kenneth J.

    2011-01-01

    Prospective associations between quantity and frequency of alcohol consumption and cancer-specific mortality were studied using a nationally representative sample with pooled data from the 1988, 1990, 1991, and 1997–2004 administrations of the National Health Interview Survey (n = 323,354). By 2006, 8,362 participants had died of cancer. Cox proportional hazards regression was used to estimate relative risks. Among current alcohol drinkers, for all-site cancer mortality, higher-quantity drinking (≥3 drinks on drinking days vs. 1 drink on drinking days) was associated with increased risk among men (relative risk (RR) = 1.24, 95% confidence interval (CI): 1.09, 1.41; P for linear trend = 0.001); higher-frequency drinking (≥3 days/week vs. cancer mortality results were similar, but among never smokers, results were null. For colorectal cancer mortality, higher-quantity drinking was associated with increased risk among women (RR = 1.93, 95% CI: 1.17, 3.18; P-trend = 0.03). Higher-frequency drinking was associated with increased risk of prostate cancer (RR = 1.55, 95% CI: 1.01, 2.38; P for quadratic effect = 0.03) and tended to be associated with increased risk of breast cancer (RR = 1.44, 95% CI: 0.96, 2.17; P-trend = 0.06). Epidemiologic studies of alcohol and cancer mortality should consider the independent effects of quantity and frequency. PMID:21965184

  4. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Directory of Open Access Journals (Sweden)

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  5. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Science.gov (United States)

    Jenkins, Jeremy L; Dean, Donald H

    2001-01-01

    Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

  6. Effect of long-term propranolol administration on specific binding of 3H-WB-4101 with rat mesenteric vascular membranes

    International Nuclear Information System (INIS)

    Ismailov, S.I.; Rozhanets, V.V.; Val'dman, A.V.

    1985-01-01

    The aim of this investigation was, first, to study the affinity of certain beta-adrenoblockers for specific binding sites of 3 H-WB-4101 (identifiable as alpha-adrenoreceptors) of brain membranes and, second, to study the characteristics of these same receptors in membranes of mesenteric vessels of rats during long-term administration of propranolol. Isotherms of specific binding, because of the limited quantity of vascular membranes, were determined by the use of three concentrations of 3 H-WB-4101: 0.1, 0.5, and 1.0 nM. It is shown that some beta-adrenoblockers have weak affinity for alpha-adrenoreceptors of brain synaptic membranes exhibited only when these compounds are present in relatively high concentrations. It is also shown that administration of propranolol for 15 days led to a significant decrease in affinity of the alpha-adrenorecptors for their specific antagonist WB-4101

  7. Is the population level link between drinking and harm similar for women and men?--a time series analysis with focus on gender-specific drinking and alcohol-related hospitalizations in Sweden.

    Science.gov (United States)

    Engdahl, Barbro; Ramstedt, Mats

    2011-08-01

    A question that has not been addressed in the literature is whether the population level association between alcohol and harm differs between men and women. The main aim of this article is to fill this gap by analysing recently collected time series data of male and female self-reported drinking in relation to gender-specific harm indicators in Sweden. Male and female per capita and risk consumption was estimated on the basis of self-reported data from monthly alcohol surveys for the period 2002-07. Overall per capita consumption including recorded sales and estimates of unrecorded consumption were also collected for the same period. Alcohol-related hospitalizations were used as indicators of alcohol-related harm. Data were aggregated into quarterly observations and analysed by means of time series analyses (ARIMA-modelling). Overall per capita consumption was significantly related to both male and female alcohol-related hospitalizations. Male per capita consumption and risk consumption were also significantly related to alcohol-related hospitalizations among men. Female per capita consumption and risk consumption had also a positive association with alcohol-related hospitalizations but statistical significance was only reached for alcohol poisonings where the association was even stronger than for men. Changes in alcohol consumption in Sweden was associated with changes in male and female alcohol-related hospitalizations also in analyses based on gender-specific consumption measures. There was no clear evidence that the population level association between alcohol and harm differed between men and women.

  8. Binding of the biogenic polyamines to deoxyribonucleic acids of varying base composition: base specificity and the associated energetics of the interaction.

    Science.gov (United States)

    Kabir, Ayesha; Suresh Kumar, Gopinatha

    2013-01-01

    The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results. Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines. From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.

  9. In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis.

    Science.gov (United States)

    Savory, Nasa; Lednor, Danielle; Tsukakoshi, Kaori; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Jones, Brian V; Ikebukuro, Kazunori

    2013-10-01

    Proteus mirabilis is a prominent cause of catheter-associated urinary tract infections (CAUTIs) among patients undergoing long-term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane-associated proteins in this organism. Copyright © 2013 Wiley Periodicals, Inc.

  10. The gendered trouble with alcohol: young people managing alcohol related violence.

    Science.gov (United States)

    Lindsay, Jo

    2012-05-01

    Alcohol related violence is a troubling backdrop to the social lives and relationships of many young people in post-industrial societies. The development of the night-time economy where young people are encouraged to drink heavily in entertainment precincts has increased the risk of violence. This paper reports on 60 individual structured in-depth interviews about the drinking biographies of young people (aged 20-24) living in Victoria, Australia. Twenty-six males and 34 females participated in the research. The participants discussed their experiences with alcohol over their life course to date. The material on alcohol related violence is analysed in this paper. Just over half of the participants (33/60) recounted negative experiences with alcohol related violence. The findings demonstrate the continuing gendered nature of experiences of perpetration and victimization. Participants reported that aggression and violence perpetrated by some men was fuelled by alcohol consumption and required ongoing management. Experiences of violence were also spatialized. Men were more likely to report managing and avoiding violence in particular public settings whilst more women than men discussed managing violence in domestic settings. The central argument of this paper is that incidents of alcohol related violence and reactions to it are specific gender performances that occur in specific socio-cultural contexts. In contrast to research which has found some young people enjoy the adventure and excitement of alcohol related violence the mainstream participants in this study saw violence as a negative force to be managed and preferably avoided. Understanding violence as a dynamic gender performance complicates the development of policy measures designed to minimize harm but also offers a more holistic approach to developing effective policy in this domain. There is a need for greater acknowledgement that alcohol related violence in public venues and in families is primarily about

  11. Specific in vivo binding of 3H-spiperone to individual lobes of the pituitary gland of the rat. Evidence for the labelling of dopamine receptors

    International Nuclear Information System (INIS)

    Koehler, C.; Fahlberg, K.

    1985-01-01

    The in vivo binding of 3 H-spiperone to individual lobes of the pituitary gland was studied after intravenous injections in unanesthetized male rats. The binding was found to be saturable and reversible. The percentage of total binding of 3 H-spiperone that was specific binding was highest in the intermediate (approx= 75%) and lowest in the posterior (approx= 35%) lobes. The regional distribution of 3 H-spiperone binding 1 hour after injections was the following: intermediate>anterior>posterior. Pharmacological analysis of the in vivo 3 H-spiperone binding showed that dopamine agonists (e.g. bromocriptine, N-n-propylnorapomorphine) and antagonsits could prevent the in vivo binding of sup3H-spiperone in all three parts of the gland. The substituted benzamide drugs remoxipride and raclopride blocked the in vivo 3 H-spiperone binding in the anterior and intermediate lobes but did not reduce the 3 H-spiperone binding in the posterior part, except when given in very high doses. Taken together, the present study has shown that 3 H-spiperone can be used in studies of the dopamine receptors in the anterior, intermediate and posterior lobes of the pituitary gland, but the proportion of non-specific binding is higher than in the striatum. The use of in vivo 3 H-spiperone binding may thus be a useful method to study the regulation and pharmacology of these receptors in situ. (Author)

  12. CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function.

    Science.gov (United States)

    Washington, Shannan D; Musarrat, Farhana; Ertel, Monica K; Backes, Gregory L; Neumann, Donna M

    2018-04-15

    There are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner. IMPORTANCE The role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1. Copyright © 2018 American Society for Microbiology.

  13. Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

    Science.gov (United States)

    Vippagunta, S R; Dorn, A; Matile, H; Bhattacharjee, A K; Karle, J M; Ellis, W Y; Ridley, R G; Vennerstrom, J L

    1999-11-04

    Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association

  14. Simplified immunoassay for rapid Dengue serotype diagnosis, revealing insensitivity to non-specific binding interference

    Directory of Open Access Journals (Sweden)

    Fernanda C.C.L. Loureiro

    2017-04-01

    Full Text Available Proof of concept of an immunoassay, which is easy to implement, for rapid Dengue virus (DENV serotype diagnosis, in the early infection stage, is reported. The four-layer assay is immobilized onto a thin gold film and relies on a low cost, disposable polymer biochip for optical surface plasmon resonance sensing and detection. The protocol comprises Neutravidin-Biotin mediated monoclonal antibody (MAB attachment as the functionalized sensing element. Formation of the MAB-DENV complex results in a pronounced thickness change that is optically recorded in real time, employing a microfluidic set-up. Virus presence is confirmed by atomic force microscopy from the same sample. Serum samples were collected from a patient in acute febrile state. Simultaneous serological analysis by means of the reverse transcription polymerase chain reaction, independently, confirmed presence of DENV2 and DENV3. The protocol proved applicable in presence of strong non-specific binding interference that originates from, and is caused by, various blood, serum and other body fluid constituents. False positive indications for both, negative serum and blood control samples were not observed. The achievable limit of detection was estimated to be 2×104 particles/ml. Eventually, the method can be modified towards detection of other viruses by using the same protocol. Keywords: Immuno-assay, Dengue virus detection, Non-specific binding

  15. Pharmacokinetic and pharmacodynamic drug interactions with ethanol (alcohol).

    Science.gov (United States)

    Chan, Lingtak-Neander; Anderson, Gail D

    2014-12-01

    Ethanol (alcohol) is one of the most widely used legal drugs in the world. Ethanol is metabolized by alcohol dehydrogenase (ADH) and the cytochrome P450 (CYP) 2E1 drug-metabolizing enzyme that is also responsible for the biotransformation of xenobiotics and fatty acids. Drugs that inhibit ADH or CYP2E1 are the most likely theoretical compounds that would lead to a clinically significant pharmacokinetic interaction with ethanol, which include only a limited number of drugs. Acute ethanol primarily alters the pharmacokinetics of other drugs by changing the rate and extent of absorption, with more limited effects on clearance. Both acute and chronic ethanol use can cause transient changes to many physiologic responses in different organ systems such as hypotension and impairment of motor and cognitive functions, resulting in both pharmacokinetic and pharmacodynamic interactions. Evaluating drug interactions with long-term use of ethanol is uniquely challenging. Specifically, it is difficult to distinguish between the effects of long-term ethanol use on liver pathology and chronic malnutrition. Ethanol-induced liver disease results in decreased activity of hepatic metabolic enzymes and changes in protein binding. Clinical studies that include patients with chronic alcohol use may be evaluating the effects of mild cirrhosis on liver metabolism, and not just ethanol itself. The definition of chronic alcohol use is very inconsistent, which greatly affects the quality of the data and clinical application of the results. Our study of the literature has shown that a significantly higher volume of clinical studies have focused on the pharmacokinetic interactions of ethanol and other drugs. The data on pharmacodynamic interactions are more limited and future research addressing pharmacodynamic interactions with ethanol, especially regarding the non-central nervous system effects, is much needed.

  16. A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.P.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

    2004-01-01

    Homology modeling of gastric H, K-ATPase based on the E-2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791)

  17. A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase.

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.P.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

    2004-01-01

    Homology modeling of gastric H,K-ATPase based on the E2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E2 form-specific salt bridge between Glu820 (M6) and Lys791 (M5).

  18. Asymmetric Reduction of Substituted 2-Tetralones by Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase

    KAUST Repository

    Bsharat, Odey; Musa, Musa M.; Vieille, Claire; Oladepo, Sulayman; Takahashi, Masateru; Hamdan, Samir

    2017-01-01

    Ketones bearing two bulky substituents, named bulky-bulky ketones, were successfully reduced to their corresponding optically enriched alcohols by using various mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Substituted 2-tetralones, in particular, were reduced to 2-tetralols with high conversion and high enantioselectivity. The pharmacological importance of substituted 2-tetralols as key drug-building blocks makes our biocatalytic reduction method a highly essential tool. We showed that changing the position of the substituent on the aromatic ring of 2-tetralones impacts their binding affinity and the reaction maximum catalytic rate. Docking studies with several TeSADH mutants explain how the position of the substituent on the tetralone influences the binding orientation of substituted 2-tetralones and their reaction stereoselectivity.

  19. Asymmetric Reduction of Substituted 2-Tetralones by Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase

    KAUST Repository

    Bsharat, Odey

    2017-01-30

    Ketones bearing two bulky substituents, named bulky-bulky ketones, were successfully reduced to their corresponding optically enriched alcohols by using various mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Substituted 2-tetralones, in particular, were reduced to 2-tetralols with high conversion and high enantioselectivity. The pharmacological importance of substituted 2-tetralols as key drug-building blocks makes our biocatalytic reduction method a highly essential tool. We showed that changing the position of the substituent on the aromatic ring of 2-tetralones impacts their binding affinity and the reaction maximum catalytic rate. Docking studies with several TeSADH mutants explain how the position of the substituent on the tetralone influences the binding orientation of substituted 2-tetralones and their reaction stereoselectivity.

  20. Alcohol-Induced Blackout

    Directory of Open Access Journals (Sweden)

    Dai Jin Kim

    2009-11-01

    Full Text Available For a long time, alcohol was thought to exert a general depressant effect on the central nervous system (CNS. However, currently the consensus is that specific regions of the brain are selectively vulnerable to the acute effects of alcohol. An alcohol-induced blackout is the classic example; the subject is temporarily unable to form new long-term memories while relatively maintaining other skills such as talking or even driving. A recent study showed that alcohol can cause retrograde memory impairment, that is, blackouts due to retrieval impairments as well as those due to deficits in encoding. Alcoholic blackouts may be complete (en bloc or partial (fragmentary depending on severity of memory impairment. In fragmentary blackouts, cueing often aids recall. Memory impairment during acute intoxication involves dysfunction of episodic memory, a type of memory encoded with spatial and social context. Recent studies have shown that there are multiple memory systems supported by discrete brain regions, and the acute effects of alcohol on learning and memory may result from alteration of the hippocampus and related structures on a cellular level. A rapid increase in blood alcohol concentration (BAC is most consistently associated with the likelihood of a blackout. However, not all subjects experience blackouts, implying that genetic factors play a role in determining CNS vulnerability to the effects of alcohol. This factor may predispose an individual to alcoholism, as altered memory function during intoxication may affect an individual‟s alcohol expectancy; one may perceive positive aspects of intoxication while unintentionally ignoring the negative aspects. Extensive research on memory and learning as well as findings related to the acute effects of alcohol on the brain may elucidate the mechanisms and impact associated with the alcohol- induced blackout.

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

    Science.gov (United States)

    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

  2. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  3. Specific binding of an immunoreactive and biologically active 125I-labeled substance P derivative to mouse mesencephalic cells in primary culture

    International Nuclear Information System (INIS)

    Beaujouan, J.C.; Torrens, Y.; Herbet, A.; Daguet, M.C.; Glowinski, J.; Prochiantz, A.

    1982-01-01

    Binding characteristics of 125 I-labeled Bolton-Hunter substance P ([ 125 I]BHSP), a radioactive analogue of substance P, were studied with mesencephalic primary cultures prepared from embryonic mouse brain. Nonspecific binding represented no more than 20% of the total binding observed on the cells. In contrast, significant specific binding--saturable, reversible, and temperature-dependent--was demonstrated. Scatchard analysis of concentration-dependent binding saturation indicates a single population of noninteracting sites with a high affinity (Kd . 169 pM). Substance P and different substance P analogues were tested for their competitive potencies with regard to [ 125 I]BHSP binding. BHSP itself, substance P, (Tyr8)-substance P, and (nor-Leu11)-substance P strongly inhibited the binding. Good inhibition was also obtained with physalaemin and eledoisin, two peptides structurally related to substance P. When substance P C-terminal fragments were tested for their ability to compete with [ 125 I]BHSP binding, a good relationship was found between competitive activity and peptide length. Regional distribution of [ 125 I]BHSP binding sites was found using primary cultures obtained from different regions of embryonic mouse brain. Mesencephalic, hypothalamic, and striatal cultures had the highest [ 125 I]BHSP binding capacities, whereas cortical, hippocampal, and cerebellar cells shared only little binding activity. Finally, when mesencephalic cells were grown under conditions impairing glial development, [ 125 I]BHSP binding was not affected, demonstrating that binding sites are located on neuronal cells

  4. Alcohol consumption and sport: a cross-sectional study of alcohol management practices associated with at-risk alcohol consumption at community football clubs.

    Science.gov (United States)

    Kingsland, Melanie; Wolfenden, Luke; Rowland, Bosco C; Gillham, Karen E; Kennedy, Vanessa J; Ramsden, Robyn L; Colbran, Richard W; Weir, Sarah; Wiggers, John H

    2013-08-16

    Excessive alcohol consumption is responsible for considerable harm from chronic disease and injury. Within most developed countries, members of sporting clubs participate in at-risk alcohol consumption at levels above that of communities generally. There has been limited research investigating the predictors of at-risk alcohol consumption in sporting settings, particularly at the non-elite level. The purpose of this study was to examine the association between the alcohol management practices and characteristics of community football clubs and at-risk alcohol consumption by club members. A cross sectional survey of community football club management representatives and members was conducted. Logistic regression analysis (adjusting for clustering by club) was used to determine the association between the alcohol management practices (including alcohol management policy, alcohol-related sponsorship, availability of low- and non-alcoholic drinks, and alcohol-related promotions, awards and prizes) and characteristics (football code, size and location) of sporting clubs and at-risk alcohol consumption by club members. Members of clubs that served alcohol to intoxicated people [OR: 2.23 (95% CI: 1.26-3.93)], conducted 'happy hour' promotions [OR: 2.84 (95% CI: 1.84-4.38)] or provided alcohol-only awards and prizes [OR: 1.80 (95% CI: 1.16-2.80)] were at significantly greater odds of consuming alcohol at risky levels than members of clubs that did not have such alcohol management practices. At-risk alcohol consumption was also more likely among members of clubs with less than 150 players compared with larger clubs [OR:1.45 (95% CI: 1.02-2.05)] and amongst members of particular football codes. The findings of this study suggest a need and opportunity for the implementation of alcohol harm reduction strategies targeting specific alcohol management practices at community football clubs.

  5. Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

    Science.gov (United States)

    Peterson, R C; Reich, M F; Dunn, P E; Law, J H; Katzenellnbogen, J A

    1977-05-17

    A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

  6. Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β-Lactamases.

    Science.gov (United States)

    Hargis, Jacqueline C; Vankayala, Sai Lakshmana; White, Justin K; Woodcock, H Lee

    2014-02-11

    Bacterial resistance to standard (i.e., β-lactam-based) antibiotics has become a global pandemic. Simultaneously, research into the underlying causes of resistance has slowed substantially, although its importance is universally recognized. Key to unraveling critical details is characterization of the noncovalent interactions that govern binding and specificity (DD-peptidases, antibiotic targets, versus β-lactamases, the evolutionarily derived enzymes that play a major role in resistance) and ultimately resistance as a whole. Herein, we describe a detailed investigation that elicits new chemical insights into these underlying intermolecular interactions. Benzylpenicillin and a novel β-lactam peptidomimetic complexed to the Stremptomyces R61 peptidase are examined using an arsenal of computational techniques: MD simulations, QM/MM calculations, charge perturbation analysis, QM/MM orbital analysis, bioinformatics, flexible receptor/flexible ligand docking, and computational ADME predictions. Several key molecular level interactions are identified that not only shed light onto fundamental resistance mechanisms, but also offer explanations for observed specificity. Specifically, an extended π-π network is elucidated that suggests antibacterial resistance has evolved, in part, due to stabilizing aromatic interactions. Additionally, interactions between the protein and peptidomimetic substrate are identified and characterized. Of particular interest is a water-mediated salt bridge between Asp217 and the positively charged N-terminus of the peptidomimetic, revealing an interaction that may significantly contribute to β-lactam specificity. Finally, interaction information is used to suggest modifications to current β-lactam compounds that should both improve binding and specificity in DD-peptidases and their physiochemical properties.

  7. A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

    Science.gov (United States)

    Stewart, James J P

    2016-11-01

    A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

  8. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  9. Associations between bar patron alcohol intoxication and tobacco smoking.

    Science.gov (United States)

    Rossheim, Matthew E; Thombs, Dennis L; O'Mara, Ryan J; Bastian, Nicholas; Suzuki, Sumihiro

    2013-11-01

    To examine the event-specific relationship between alcohol intoxication and nighttime tobacco smoking among college bar patrons. In this secondary analysis of existing data, we examined event-specific associations between self-report measures of tobacco smoking and breath alcohol concentration (BrAC) readings obtained from 424 patrons exiting on-premise drinking establishments. In a multivariable logistic regression analysis, acute alcohol intoxication was positively associated with same-night incidents of smoking tobacco, adjusting for the effects of established smoking practices and other potential confounders. This investigation is the first known study using data collected in an on-premise drinking setting to link alcohol intoxication to specific incidents of tobacco smoking.

  10. Neuronal nicotinic acetylcholine receptors: Common molecular substrates of nicotine and alcohol dependence

    Directory of Open Access Journals (Sweden)

    Linzy M. Hendrickson

    2013-04-01

    Full Text Available Alcohol and nicotine are often co-abused. As many as 80-95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs, ligand-gated cation channels normally activated by endogenous acetylcholine (ACh, ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA which project to the nucleus accumbens (NAc. Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from preclinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence.

  11. SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

    International Nuclear Information System (INIS)

    Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

    2007-01-01

    Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

  12. A single, specific thymine mutation in the ComK-Binding site severely decreases binding and transcription activation by the competence transcription factor ComK of Bacillus subtilis

    NARCIS (Netherlands)

    Susanna, Kim A.; Mironczuk, Aleksandra M.; Smits, Wiep Klaas; Hamoen, Leendert W.; Kuipers, Oscar P.

    The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region.

  13. There has been little progress in implementing comprehensive alcohol control strategies in Africa.

    Science.gov (United States)

    Peer, Nasheeta

    2017-11-01

    Alcohol is the most common substance of addiction and a threat not only to health but also to sustainable human development. Consequently, at least a 10% relative reduction in the harmful use of alcohol has been advocated by the World Health Organization (WHO). This perspective describes alcohol use in Africa, strategies to reduce harmful alcohol use, and the ability of African countries to meet this target. Although alcohol consumption in Africa was intermediate compared to other world regions, the total alcohol per capita among alcohol consumers was the second highest (19.5 liters); 19% of Sub-Saharan African men could be classified as binge drinkers. The alcohol industry is the key driver behind the uptake of alcohol use and misuse. The most cost-effective ways to reduce alcohol-related harm is to make alcohol less available and more expensive and to prohibit alcohol advertising. Most African countries have alcohol excise taxes, but these are not adjusted for inflation, meaning that the effectiveness of these taxes will likely decrease with time, leading to greater affordability. The majority of African countries do not have legally binding regulations for alcohol marketing. Alcohol misuse in Africa is not being addressed at a time when available strategies can efficiently and cost-effectively control alcohol-related harm.

  14. Examining alcohol consumption with the theory of planned behaviour: Do health and alcohol knowledge play a role?

    Science.gov (United States)

    Hasking, Penelope; Schofield, Lachlan

    2015-01-01

    We used the theory of planned behaviour (TPB) to investigate factors associated with alcohol consumption among university students, and to examine whether general or alcohol-specific health knowledge acts as a moderator in the relationship between elements of the theory and drinking behaviour. Participants were 258 Australian undergraduate university students (79% female) who completed an online questionnaire, assessing the constructs of interest. The hypothesis that intentions and behaviour would be successfully predicted using the theory was generally supported. Little evidence for the moderating effect of knowledge on the TPB variables was observed, although both general and alcohol-specific health knowledge moderated the relationship between intentions and behaviours. Contrary to expectation, more accurate knowledge strengthened this relationship. Further work is necessary to investigate the role of knowledge in limiting alcohol-related harms.

  15. Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

    Science.gov (United States)

    Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

    2015-01-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

  16. Concurrent and Simultaneous Drug and Alcohol Use: Results of the 2000 National Alcohol Survey

    OpenAIRE

    Midanik, Lorraine T.; Tam, Tammy W.; Weisner, Constance

    2007-01-01

    This study estimates the prevalence, assesses predictors and evaluates factors associated with concurrent and simultaneous use of drugs and alcohol in the United States population. Using data from the 2000 National Alcohol Survey (n = 7,612), respondents were asked if they used specific drugs in the last 12 months. Current drinkers who reported using each type of drug were asked if they used alcohol and the drug at the same time. Approximately 10% reported using marijuana in the last 12 month...

  17. Revisiting the link between body and agency: visual movement congruency enhances intentional binding but is not body-specific.

    Science.gov (United States)

    Zopf, Regine; Polito, Vince; Moore, James

    2018-01-09

    Embodiment and agency are key aspects of how we perceive ourselves that have typically been associated with independent mechanisms. Recent work, however, has suggested that these mechanisms are related. The sense of agency arises from recognising a causal influence on the external world. This influence is typically realised through bodily movements and thus the perception of the bodily self could also be crucial for agency. We investigated whether a key index of agency - intentional binding - was modulated by body-specific information. Participants judged the interval between pressing a button and a subsequent tone. We used virtual reality to manipulate two aspects of movement feedback. First, form: participants viewed a virtual hand or sphere. Second, movement congruency: the viewed object moved congruently or incongruently with the participant's hidden hand. Both factors, form and movement congruency, significantly influenced embodiment. However, only movement congruency influenced intentional binding. Binding was increased for congruent compared to incongruent movement feedback irrespective of form. This shows that the comparison between viewed and performed movements provides an important cue for agency, whereas body-specific visual form does not. We suggest that embodiment and agency mechanisms both depend on comparisons across sensorimotor signals but that they are influenced by distinct factors.

  18. Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic, calorimetric and modeling investigations

    International Nuclear Information System (INIS)

    Wang Gongke; Yan Changling; Wang Dongchao; Li Dan; Lu Yan

    2012-01-01

    One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl) -3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10 4 M −1 . According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven. - Highlights: ► Medically important dihydropyrimidinones derivative EMMD is synthesized. ► EMMD is intercalative binding into ctDNA helix. ► Hydrogen bonding may play an essential role in the binding of EMCD with ctDNA. ► This binding interaction is predominantly enthalpy driven.

  19. Impulsivity and alcohol demand in relation to combined alcohol and caffeine use.

    Science.gov (United States)

    Amlung, Michael; Few, Lauren R; Howland, Jonathan; Rohsenow, Damaris J; Metrik, Jane; MacKillop, James

    2013-12-01

    Problematic alcohol use among college students continues to be a prominent concern in the United States, including the growing trend of consuming caffeinated alcoholic beverages (CABs). Epidemiologically, CAB use is associated with incremental risks from drinking, although these relationships could be due to common predisposing factors rather than specifically due to CABs. This study investigated the relationship between CAB use, alcohol misuse, and person-level characteristics, including impulsive personality traits, delayed reward discounting, and behavioral economic demand for alcohol use. Participants were 273 regularly drinking undergraduate students. Frequency of CAB use was assessed over the past month. A multidimensional assessment of impulsivity included the UPPS-P questionnaire, which measures positive and negative urgency, premeditation (lack thereof), perseverance (lack thereof), and sensation seeking (Lynam, Smith, Whiteside, & Cyders, 2007), and a validated questionnaire-based measure of delayed reward discounting. Demand was assessed via a hypothetical alcohol purchase task. Frequency of CAB consumption was significantly higher in men than in women and was also associated with higher impulsivity on the majority of the UPPS-P subscales, steeper delayed reward discounting, and greater demand for alcohol. Significant correlations between CAB use and both alcohol demand and lack of premeditation remained present after including level of alcohol misuse in partial correlations. In a hierarchical linear regression incorporating demographic, demand, and impulsivity variables, CAB frequency continued to be a significant predictor of hazardous alcohol use. These results suggest that although there are significant associations between CAB consumption and gender, impulsivity, and alcohol demand, CAB use continues to be associated with alcohol misuse after controlling for these variables.

  20. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  1. Binding of the biogenic polyamines to deoxyribonucleic acids of varying base composition: base specificity and the associated energetics of the interaction.

    Directory of Open Access Journals (Sweden)

    Ayesha Kabir

    Full Text Available BACKGROUND: The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results. METHODOLOGY/PRINCIPAL FINDINGS: Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines. CONCLUSION/SIGNIFICANCE: From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.

  2. [Current peculiarities of alcoholic psychosis].

    Science.gov (United States)

    Aleksin, D S; Egorov, A Iu

    2011-01-01

    The follow-up study of alcoholic psychoses in male patients admitted to a clinical department of a psychiatric hospital in 2005-2007 was carried out. Patients with alcoholic psychoses made up from 15 to 30% of all patients. The number of psychosis had seasonal variations with the elevations in spring and autumn, peaks in January, lune and October. Alcoholic delirium morbidity made up from 69 to 82% of the total number of alcoholic psychoses, alcoholic hallucinosis varied from 14 to 27%. Other forms were presented by single cases. In alcoholic delirium hallucinations had brighter, sated character. The most specific were visual hallucinations in the form of zoohallucinations, hallucinations of an oral cavity ("sensation of threads, hair etc"). The most often observable characters were "extraneous people, animal, demons". In alcoholic hallucinosis, verbal contrast hallucinations, making comment hallucinations, visual illusions were most frequent. The family history of mental disorders and alcoholism was noted in 30% of patients with alcoholic psychosis. The probability of occurrence of alcoholic psychoses depended on the quality of consumed drinks. The presence of a cranial-brain injury in the anamnesis considerably aggravated the disease forecast and increased the risk of seizure syndrome.

  3. Associations between social media displays and event-specific alcohol consumption by college students.

    Science.gov (United States)

    Moreno, Megan A; Kacvinsky, Lauren; Pumper, Megan; Wachowski, Leah; Whitehill, Jennifer M

    2013-12-01

    The Mifflin Street Block Party is a yearly Wisconsin event known for high levels of alcohol consumption and previous negative outcomes. This study investigated displayed Mifflin references on Facebook and their association with alcohol consumption at the block party. Participants included first-year college students who were enrolled in a longitudinal study involving Facebook profile assessments and interviews. We identified a subset of participants who were interviewed within 28 days following the Mifflin St Block Party. Participants were categorized as "Mifflin Displayers" or "Non-displayers" based on Facebook profile content. Interviews included the timeline follow-back method to assess alcohol use in the past 28 days. Analysis included logistic and linear regression. Among the 66 participants included in this study, 45 (68.2%) were female and 38 (50%) were Mifflin Displayers on Facebook. Among the Mifflin Displayer participants, 18 (27.2%) displayed prior to Mifflin, 11 displayed the day of Mifflin (16.7%) and 19 (28.8%) displayed after. Some participants displayed in more than 1 time frame. A total of 40 (60.6%) reported alcohol use on the day of the Mifflin Street Block Party. The mean number of drinks reported on the day of Mifflin was 8.8 (SD = 6.1), with a range of 1 to 35. Displayed references to Mifflin on Facebook were positively associated with reporting alcohol use at Mifflin (OR = 20.9, 95% CI 5.6-78.8). Displaying Facebook references to Mifflin was associated with alcohol consumption on the day of the event. Future prevention efforts could consider creating Facebook advertisements with safety messages triggered by Mifflin displays.

  4. Racial/ethnic differences in the influence of cultural values, alcohol resistance self-efficacy, and alcohol expectancies on risk for alcohol initiation.

    Science.gov (United States)

    Shih, Regina A; Miles, Jeremy N V; Tucker, Joan S; Zhou, Annie J; D'Amico, Elizabeth J

    2012-09-01

    Prior research has reported racial/ethnic differences in the early initiation of alcohol use, suggesting that cultural values that are central to specific racial/ethnic groups may be influencing these differences. This 1-year longitudinal study examines associations between two types of cultural values, parental respect (honor for one's parents) and familism (connectedness with family), both measured at baseline, and subsequent alcohol initiation in a sample of 6,054 (approximately 49% male, 57% Hispanic, 22% Asian, 18% non-Hispanic White, and 4% non-Hispanic Black) middle school students in Southern California. We tested whether the associations of cultural values with alcohol initiation could be explained by baseline measures of alcohol resistance self-efficacy (RSE) and alcohol expectancies. We also explored whether these pathways differed by race/ethnicity. In the full sample, adolescents with higher parental respect were less likely to initiate alcohol use, an association that was partially explained by higher RSE and fewer positive alcohol expectancies. Familism was not significantly related to alcohol initiation. Comparing racial/ethnic groups, higher parental respect was protective against alcohol initiation for Whites and Asians, but not Blacks or Hispanics. There were no racial/ethnic differences in the association between familism and alcohol initiation. Results suggest that cultural values are important factors in the decision to use alcohol and these values appear to operate in part, by influencing alcohol positive expectancies and RSE. Interventions that focus on maintaining strong cultural values and building strong bonds between adolescents and their families may help reduce the risk of alcohol initiation. PsycINFO Database Record (c) 2012 APA, all rights reserved.

  5. Aggression-related alcohol expectancies and barroom aggression among construction tradespeople.

    Science.gov (United States)

    Zinkiewicz, Lucy; Smith, Georgia; Burn, Michele; Litherland, Steven; Wells, Samantha; Graham, Kathryn; Miller, Peter

    2016-09-01

    Few studies have investigated the relationship of barroom aggression with both general and barroom-specific alcohol expectancies. The present study investigated these associations in a rarely studied and high-risk population: construction tradespeople. Male construction tradespeople (n = 211) aged 18-35 years (M = 21.91, SD = 4.08 years) participated in a face-to-face questionnaire assessing general and barroom-specific alcohol expectancies and perpetration of physical and verbal barroom aggression as well as control variables, age, alcohol consumption and trait aggression. Sequential logistic regression analyses revealed that general alcohol-aggression expectancies of courage or dominance were not predictive of either verbal or physical barroom aggression after controlling for age, alcohol consumption and trait aggression. However, barroom-specific alcohol expectancies were associated with both verbal and physical barroom aggression, with positive associations found for expected hyper-emotionality and protective effects for expected cognitive impairment. In a population where rates of risky drinking and barroom aggression are high, specific expectations about the effects of drinking in bars may influence subsequent aggressive behaviour in bars. [Zinkiewicz L, Smith G, Burn M, Litherland S, Wells S, Graham K, Miller P. Aggression-related alcohol expectancies and barroom aggression among construction tradespeople. Drug Alcohol Rev 2016;35:549-556]. © 2015 Australasian Professional Society on Alcohol and other Drugs.

  6. Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

    Science.gov (United States)

    Abramyan, John; Stajich, Jason E

    2012-01-01

    Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide

  7. Lignans from the roots of Urtica dioica and their metabolites bind to human sex hormone binding globulin (SHBG).

    Science.gov (United States)

    Schöttner, M; Gansser, D; Spiteller, G

    1997-12-01

    Polar extracts of the stinging nettle (Urtica dioica L.) roots contain the ligans (+)-neoolivil, (-)-secoisolariciresinol, dehydrodiconiferyl alcohol, isolariciresinol, pinoresinol, and 3,4-divanillyltetrahydrofuran. These compounds were either isolated from Urtica roots, or obtained semisynthetically. Their affinity to human sex hormone binding globulin (SHBG) was tested in an in vitro assay. In addition, the main intestinal transformation products of plant lignans in humans, enterodiol and enterolactone, together with enterofuran were checked for their activity. All lignans except (-)-pinoresinol developed a binding affinity to SHBG in the in vitro assay. The affinity of (-)-3,4-divanillyltetrahydrofuran was outstandingly high. These findings are discussed with respect to potential beneficial effects of plant lignans on benign prostatic hyperplasia (BPH).

  8. Alcoholism and Alcohol Abuse

    Science.gov (United States)

    ... their drinking causes distress and harm. It includes alcoholism and alcohol abuse. Alcoholism, or alcohol dependence, is a disease that causes ... the liver, brain, and other organs. Drinking during pregnancy can harm your baby. Alcohol also increases the ...

  9. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

    Science.gov (United States)

    Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

    1994-12-20

    The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

  10. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  11. Impact of a new alcohol policy on homemade alcohol consumption and sales in Russia.

    Science.gov (United States)

    Radaev, Vadim

    2015-05-01

    To describe the effects of Russian policy since 2006 affecting price and availability on the consumption of recorded and unrecorded alcohol, with specific reference to homemade alcohol, and to investigate other factors affecting homemade alcohol consumption and purchasing. Consumption and preferred beverage data were collected from RLMS-HSE nationwide panel surveys from 1994 to 2013, with a detailed analysis of 2012 data (18,221 respondents aged 16+ years). Official statistics on manufactured alcohol sales, regional price increase and real disposable income were used. Homemade distilled spirits (samogon) consumption decreased together with that of recorded and unrecorded manufactured spirits since 2000. The consumption of spirits was partially replaced by the consumption of beer and wine. These trends in alcohol consumption were interrupted in 2008-2013. The interruption was more likely affected by the economic crisis and recession than by the new alcohol policy. Social networks and availability of unrecorded alcohol were more important predictors of homemade alcohol consumption and purchasing than was a recorded alcohol price increase. Homemade alcohol consumption does not replace the declining market for recorded spirits in Russia. The effects of economic and social factors on homemade alcohol consumption are greater than are the short-term effects of the new alcohol policy. The very recent (2015) reduction of the minimum unit price of vodka may be premature. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  12. The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

    Directory of Open Access Journals (Sweden)

    Helene J Bustad

    Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

  13. The epigenetic landscape of alcoholism.

    Science.gov (United States)

    Krishnan, Harish R; Sakharkar, Amul J; Teppen, Tara L; Berkel, Tiffani D M; Pandey, Subhash C

    2014-01-01

    Alcoholism is a complex psychiatric disorder that has a multifactorial etiology. Epigenetic mechanisms are uniquely capable of accounting for the multifactorial nature of the disease in that they are highly stable and are affected by environmental factors, including alcohol itself. Chromatin remodeling causes changes in gene expression in specific brain regions contributing to the endophenotypes of alcoholism such as tolerance and dependence. The epigenetic mechanisms that regulate changes in gene expression observed in addictive behaviors respond not only to alcohol exposure but also to comorbid psychopathology such as the presence of anxiety and stress. This review summarizes recent developments in epigenetic research that may play a role in alcoholism. We propose that pharmacologically manipulating epigenetic targets, as demonstrated in various preclinical models, hold great therapeutic potential in the treatment and prevention of alcoholism. © 2014 Elsevier Inc. All rights reserved.

  14. Alcohol under the radar: do we have policy options regarding unrecorded alcohol?

    Science.gov (United States)

    Lachenmeier, Dirk W; Taylor, Benjamin J; Rehm, Jürgen

    2011-03-01

    According to the World Health Organization, the public health impact of illicit alcohol and informally produced alcohol should be reduced. This paper summarizes and evaluates the evidence base about policy and intervention options regarding unrecorded alcohol consumption. A systematic review of the literature using electronic databases. The literature on unrecorded consumption was sparse with less than 30 articles about policy options, mostly based on observational studies. The most simplistic option to reduce unrecorded consumption would be to lower recorded alcohol prices to remove the economic incentive of buying unrecorded alcohol. However, this may increase the net total alcohol consumption, making it an unappealing public health policy option. Other policy options largely depend on the specific sub-group of unrecorded alcohol. The prohibition of toxic compounds used to denature alcohol (e.g. methanol) can improve health outcomes associated with surrogate alcohol consumption. Cross-border shopping can be reduced by either narrowing the tax differences, or stricter control. Actions limiting illegal trade and counterfeiting include introduction of tax stamps and electronic surveillance systems of alcohol trade. Education campaigns might increase the awareness about the risks associated with illegal alcohol. The most problematic category appears to be the home and small-scale artisanal production, for which the most promising option is to offer financial incentives to the producers for registration and quality control. Even though there are suggestions and theories on how to reduce unrecorded alcohol consumption, there is currently no clear evidence base on the effectiveness or cost effectiveness of available policy options. In addition, the differences in consumption levels, types of unrecorded alcohol, culture and tradition point to different measures in different parts of the world. Thus, the recommendation of a framework for moving forward in decision making

  15. Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

    Science.gov (United States)

    Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

    2014-08-01

    Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

  16. Alcoholic liver disease in Nepal: identifying homemade alcohol as a culprit.

    Science.gov (United States)

    Pradhan, Bickram; Hadengue, Antoine; Chappuis, François; Chaudhary, Shatdal; Baral, Dharanidhar; Gache, Pascal; Karki, Prahlad; Rijal, Suman

    2015-01-01

    Though the type of alcohol consumed is not thought to be associated with alcoholic liver disease (ALD), some studies have shown a beverage-specific effect. In the present study, we aim to study the effects of locally brewed alcoholic beverages on the development of liver disease. This cross-sectional study was conducted at the internal medicine department of a university hospital in Nepal. All patients classified as having either alcohol abuse or alcohol dependence by the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition were evaluated for the presence of ALD. A total of 1,500 patients were screened, of which, 447 patients had ALD. Chronic liver disease (CLD) was detected in 144 patients (9.6%). Most of the patients consumed homemade locally brewed alcohol. On multivariate analysis, the following variables were found to be significantly associated with CLD: male sex (odds ratio [OR]: 1.81; 95% confidence interval [CI]: 1.12-2.94; P=0.02): rakshi consumption ≥30 units (OR: 2.53; 95% CI: 1.07-6.01; P=0.04); and tongba consumption (OR: 3.02; 95% CI: 1.22-7.50; P=0.02). There was a significant increase in the risk of developing ALD with the consumption of rakshi and tongba after adjusting for total units consumed. The absence of striking differences between our patients with CLD and non-CLD patients with regards to the amount of alcohol consumed demonstrates that, although alcohol consumption is a prerequisite for the development of ALD, other factors like type of alcoholic beverage consumed may be involved.

  17. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    NARCIS (Netherlands)

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  18. Pharmacological modulation of mGluR7 with AMN082 and MMPIP exerts specific influences on alcohol consumption and preference in rats.

    Science.gov (United States)

    Bahi, Amine; Fizia, Katharina; Dietz, Monika; Gasparini, Fabrizio; Flor, Peter J

    2012-03-01

    Growing evidence supports a role for the central nervous system (CNS) neurotransmitter L-glutamate and its metabotropic receptors (mGluRs) in drug addiction in general and alcohol-use disorders in particular. Alcohol dependence, for instance, has a genetic component, and the recent discovery that variations in the gene coding for mGluR7 modulate alcohol consumption further validates involvement of the L-glutamate system. Consequently, increasing interest emerges in developing L-glutamatergic therapies for the treatment of alcohol abuse and dependence. To this end, we performed a detailed behavioral pharmacology study to investigate the regulation of alcohol consumption and preference following administration of the mGluR7-selective drugs N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) and 6-(4-Methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one hydrochloride (MMPIP). Upon administration of the allosteric agonist AMN082 (10 mg/kg, i.p.) in rats, there was a significant decrease in ethanol consumption and preference, without affecting ethanol blood metabolism. In contrast, mGluR7 blockade with MMPIP (10 mg/kg, i.p.) showed an increase in alcohol intake and reversed AMN082's effect on ethanol consumption and preference. Both mGluR7-directed pharmacological tools had no effect on total fluid intake, taste preference, or on spontaneous locomotor activity. In conclusion, these findings support a specific regulatory role for mGluR7 on alcohol drinking and preference and provide evidence for the use of AMN082-type drugs as potential new treatments for alcohol-use disorders in man. © 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.

  19. Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules

    DEFF Research Database (Denmark)

    Troelsen, J T; Mitchelmore, C; Sjöström, H

    1994-01-01

    Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are enterocyte-specific gene products. The identification of regulatory cis-elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal-specific nuclear factors (NF-LPH1...... and SIF1-BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis-elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 k......Da oligomeric complexes. Based on these results NF-LPH1 and SIF1-BP are suggested to be either identical or closely related molecules....

  20. Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

    Science.gov (United States)

    Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

    2017-01-01

    Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

  1. The effects of cocaine, alcohol and cocaine/alcohol combinations in conditioned taste aversion learning.

    Science.gov (United States)

    Busse, Gregory D; Verendeev, Andrey; Jones, Jermaine; Riley, Anthony L

    2005-09-01

    We have recently reported that alcohol attenuates cocaine place preferences. Although the basis for this effect is unknown, alcohol may attenuate cocaine reward by potentiating its aversive effects. To examine this possibility, these experiments assessed the effects of alcohol on cocaine-induced taste aversions under conditions similar to those that resulted in attenuated place preferences. Specifically, Experiments 1 and 2 assessed the effects of alcohol (0.5 g/kg) on taste aversions induced by 20, 30 and 40 mg/kg cocaine. Experiment 3 examined the role of intertrial interval in the effects of alcohol (0.5 g/kg) on cocaine (30 mg/kg) taste aversions. In Experiments 1 and 2, cocaine was effective at conditioning aversions. Alcohol produced no measurable effect. Combining cocaine and alcohol produced no greater aversion than cocaine alone (and, in fact, weakened aversions at the lowest dose of cocaine). In Experiment 3, varying the intertrial interval from 3 days (as in the case of Experiments 1 and 2) to 1 day (a procedure identical to that in which alcohol attenuated cocaine place preferences) resulted in significant alcohol- and cocaine-induced taste aversions. Nonetheless, alcohol remained ineffective in potentiating cocaine aversions. Thus, under these conditions alcohol does not potentiate cocaine's aversiveness. These results were discussed in terms of their implication for the effects of alcohol on cocaine-induced place preferences. Further, the effects of alcohol on place preferences conditioned by cocaine were discussed in relation to other assessments of the effects of alcohol on the affective properties of cocaine and the implications of these interactions for alcohol and cocaine co-use.

  2. Site-specific binding of a water molecule to the sulfa drugs sulfamethoxazole and sulfisoxazole: a laser-desorption isomer-specific UV and IR study.

    Science.gov (United States)

    Uhlemann, Thomas; Seidel, Sebastian; Müller, Christian W

    2018-03-07

    To determine the preferred water molecule binding sites of the polybasic sulfa drugs sulfamethoxazole (SMX) and sulfisoxazole (SIX), we have studied their monomers and monohydrated complexes through laser-desorption conformer-specific UV and IR spectroscopy. Both the SMX and SIX monomer adopt a single conformer in the molecular beam. On the basis of their conformer-specific IR spectra in the NH stretch region, these conformers were assigned to the SMX and SIX global minimum structures, both exhibiting a staggered sulfonamide group and an intramolecular C-HO[double bond, length as m-dash]S hydrogen bond. The SMX-H 2 O and SIX-H 2 O complexes each adopt a single isomer in the molecular beam. Their isomeric structures were determined based on their isomer-specific IR spectra in the NH/OH stretch region. Quantum Theory of Atoms in Molecules analysis of the calculated electron densities revealed that in the SMX-H 2 O complex the water molecule donates an O-HN hydrogen bond to the heterocycle nitrogen atom and accepts an N-HO hydrogen bond from the sulfonamide NH group. In the SIX-H 2 O complex, however, the water molecule does not bind to the heterocycle but instead donates an O-HO[double bond, length as m-dash]S hydrogen bond to the sulfonamide group and accepts an N-HO hydrogen bond from the sulfonamide NH group. Both water complexes are additionally stabilized by a C ph -HOH 2 hydrogen bond. Interacting Quantum Atoms analysis suggests that all intermolecular hydrogen bonds are dominated by the short-range exchange-correlation contribution.

  3. Alcohol demand and risk preference.

    Science.gov (United States)

    Dave, Dhaval; Saffer, Henry

    2008-12-01

    Both economists and psychologists have studied the concept of risk preference. Economists categorize individuals as more or less risk-tolerant based on the marginal utility of income. Psychologists categorize individuals' propensity towards risk based on harm avoidance, novelty seeking and reward dependence traits. The two concepts of risk are related, although the instruments used for empirical measurement are quite different. Psychologists have found risk preference to be an important determinant of alcohol consumption; however economists have not included risk preference in studies of alcohol demand. This is the first study to examine the effect of risk preference on alcohol consumption in the context of a demand function. The specifications employ multiple waves from the Panel Study of Income Dynamics (PSID) and the Health and Retirement Study (HRS), which permit the estimation of age-specific models based on nationally representative samples. Both of these data sets include a unique and consistent survey instrument designed to directly measure risk preference in accordance with the economist's definition. This study estimates the direct impact of risk preference on alcohol demand and also explores how risk preference affects the price elasticity of demand. The empirical results indicate that risk preference has a significant negative effect on alcohol consumption, with the prevalence and consumption among risk-tolerant individuals being 6-8% higher. Furthermore, the tax elasticity is similar across both risk-averse and risk-tolerant individuals. This suggests that tax policies are as equally effective in deterring alcohol consumption among those who have a higher versus a lower propensity for alcohol use.

  4. General odorant-binding proteins and sex pheromone guide larvae of Plutella xylostella to better food.

    Science.gov (United States)

    Zhu, Jiao; Ban, Liping; Song, Li-Mei; Liu, Yang; Pelosi, Paolo; Wang, Guirong

    2016-05-01

    Olfaction of Lepidopteran larvae has received little attention, compared to the damage to crops done by insects at this stage. We report that larvae of the diamondback moth Plutella xylostella are attracted to their natural sex pheromone and to their major component (Z)-11-hexadecenal, but only in a food context. For such task they use two general odorant-binding proteins (GOBPs), abundantly expressed in the three major sensilla basiconica of the larval antenna, as shown by whole-mount immunostaining and immunocytochemistry experiments. None of the three genes encoding pheromone-binding proteins (PBPs) are expressed at this stage. Both recombinant GOBPs bind (Z)-11-hexadecenal and the corresponding alcohol, but not the acetate. Binding experiments performed with five mutants of GOBP2, where aromatic residues in the binding pocket were replaced with leucine showed that only one or two amino acid substitutions can completely abolish binding to the pheromone shifting the affinity to plant-derived compounds. We hypothesise that detection of their species-specific pheromone may direct larvae to the sites of foraging chosen by their mother when laying eggs, to find better food, as well as to reduce competition with individuals of the same or other species sharing the same host plant. We also provide evidence that GOBP2 is a narrowly tuned binding protein, whose affinity can be easily switched from linear pheromones to branched plants terpenoids, representing a tool better suited for the simple olfactory system of larvae, as compared to the more sophisticated organ of adults. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Alcohol segment-specific associations between the quality of the parent-child relationship and adolescent alcohol use

    NARCIS (Netherlands)

    Mathijssen, J.J.P.; Janssen, M.M.; van Bon, M.J.H.; van Oers, J.A.M.; de Boer, E.

    2014-01-01

    Background There is much evidence that parents have an influence on the alcohol use of their children. However, in general the relationship is rather weak. A reason for this small association may be due to the fact that adolescents are a heterogeneous group and that, consequently, the association

  6. Aerobic oxidation of alcohols in visible light on Pd-grafted Ti cluster

    Science.gov (United States)

    The titanium cluster with the reduced band gap has been synthesized having the palladium nanoparticles over the surface, which not only binds to the atmospheric oxygen but also catalyzes the oxidation of alcohols under visible light.

  7. Mood, anxiety, and alcohol use disorders and later cause-specific sick leave in young adult employees.

    Science.gov (United States)

    Torvik, Fartein Ask; Reichborn-Kjennerud, Ted; Gjerde, Line C; Knudsen, Gun Peggy; Ystrom, Eivind; Tambs, Kristian; Røysamb, Espen; Østby, Kristian; Ørstavik, Ragnhild

    2016-08-03

    Mental disorders strongly influence work capability in young adults, but it is not clear which disorders that are most strongly associated with sick leave, and which diagnoses that are stated on the sick leave certificates. Better knowledge of the impairments associated with different mental disorders is needed for optimal planning of interventions and prioritization of health services. In the current study, we investigate the prospective associations between eight mood, anxiety, and alcohol use disorders, and later sick leave granted for mental, somatic, or any disorder. Lifetime mental disorders were assessed by structured diagnostic interviews in 2,178 young adults followed for eight years with registry data on sick leave. Relative risk ratios were estimated for the associations between each mental disorder and the different forms of sick leave. All included diagnoses were associated with later sick leave. In adjusted analyses, major depressive disorder and generalized anxiety disorder were the strongest predictors of sick leave granted for mental disorders, whereas social anxiety disorder and specific phobia were the strongest predictors of sick leave granted for somatic disorders. Specific phobia and major depressive disorder had the highest attributable fractions for all-cause sick leave. Mood and anxiety disorders constituted independent risk factors for all cause sick leave, whereas alcohol use disorders seemed to be of less importance in young adulthood. Disorders characterised by distress were most strongly associated with sick leave granted for mental disorders, whereas disorders characterised by fear primarily predicted sick leave granted for somatic conditions. A large part of all sick leave is related to specific phobia, due to the high prevalence of this disorder. The impairment associated with this common disorder may be under-acknowledged, and it could decrease work capacity among individuals with somatic disorders. This disorder has good treatment

  8. Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

    Science.gov (United States)

    Deng, W G; Ruan, K H; Du, M; Saunders, M A; Wu, K K

    2001-11-01

    Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

  9. Personality and Defense Styles: Clinical Specificities and Predictive Factors of Alcohol Use Disorder in Women.

    Science.gov (United States)

    Ribadier, Aurélien; Dorard, Géraldine; Varescon, Isabelle

    2016-01-01

    This study investigated personality traits and defense styles in order to determine clinical specificities and predictive factors of alcohol use disorders (AUDs) in women. A female sample, composed of AUD outpatients (n = 48) and a control group (n = 50), completed a sociodemographic self-report and questionnaires assessing personality traits (BFI), defense mechanisms and defense styles (DSQ-40). Comparative and correlational analyses, as well as univariate and multivariate logistic regressions, were performed. AUD women presented with higher neuroticism and lower extraversion and conscientiousness. They used less mature and more neurotic and immature defense styles than the control group. Concerning personality traits, high neuroticism and lower conscientiousness were predictive of AUD, as well as low mature, high neurotic, and immature defense styles. Including personality traits and defense styles in a logistic model, high neuroticism was the only AUD predictive factor. AUD women presented clinical specificities and predictive factors in personality traits and defense styles that must be taken into account in AUD studies. Implications for specific treatment for women are discussed.

  10. Molecular mechanisms of synaptic remodeling in alcoholism.

    Science.gov (United States)

    Kyzar, Evan J; Pandey, Subhash C

    2015-08-05

    Alcohol use and alcohol addiction represent dysfunctional brain circuits resulting from neuroadaptive changes during protracted alcohol exposure and its withdrawal. Alcohol exerts a potent effect on synaptic plasticity and dendritic spine formation in specific brain regions, providing a neuroanatomical substrate for the pathophysiology of alcoholism. Epigenetics has recently emerged as a critical regulator of gene expression and synaptic plasticity-related events in the brain. Alcohol exposure and withdrawal induce changes in crucial epigenetic processes in the emotional brain circuitry (amygdala) that may be relevant to the negative affective state defined as the "dark side" of addiction. Here, we review the literature concerning synaptic plasticity and epigenetics, with a particular focus on molecular events related to dendritic remodeling during alcohol abuse and alcoholism. Targeting epigenetic processes that modulate synaptic plasticity may yield novel treatments for alcoholism. Published by Elsevier Ireland Ltd.

  11. Maternal alcohol consumption producing fetal alcohol spectrum disorders (FASD): quantity, frequency, and timing of drinking.

    Science.gov (United States)

    May, Philip A; Blankenship, Jason; Marais, Anna-Susan; Gossage, J Phillip; Kalberg, Wendy O; Joubert, Belinda; Cloete, Marise; Barnard, Ronel; De Vries, Marlene; Hasken, Julie; Robinson, Luther K; Adnams, Colleen M; Buckley, David; Manning, Melanie; Parry, Charles D H; Hoyme, H Eugene; Tabachnick, Barbara; Seedat, Soraya

    2013-12-01

    Concise, accurate measures of maternal prenatal alcohol use are needed to better understand fetal alcohol spectrum disorders (FASD). Measures of drinking by mothers of children with specific FASD diagnoses and mothers of randomly-selected controls are compared and also correlated with physical and cognitive/behavioral outcomes. Measures of maternal alcohol use can differentiate maternal drinking associated with FASD from that of controls and some from mothers of alcohol-exposed normals. Six variables that combine quantity and frequency concepts distinguish mothers of FASD children from normal controls. Alcohol use variables, when applied to each trimester and three months prior to pregnancy, provide insight on critical timing of exposure as well. Measures of drinking, especially bingeing, correlate significantly with increased child dysmorphology and negative cognitive/behavioral outcomes in children, especially low non-verbal IQ, poor attention, and behavioral problems. Logistic regression links (p<.001) first trimester drinking (vs. no drinking) with FASD, elevating FASD likelihood 12 times; first and second trimester drinking increases FASD outcomes 61 times; and drinking in all trimesters 65 times. Conversely, a similar regression (p=.008) indicates that drinking only in the first trimester makes the birth of a child with an FASD 5 times less likely than drinking in all trimesters. There is significant variation in alcohol consumption both within and between diagnostic groupings of mothers bearing children diagnosed within the FASD continuum. Drinking measures are empirically identified and correlated with specific child outcomes. Alcohol use, especially heavy use, should be avoided throughout pregnancy. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  13. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

    Science.gov (United States)

    Bell, Thomas J; Eberwine, James

    2015-01-01

    RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

  14. Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

    Science.gov (United States)

    Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

    2015-03-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Amount of Televised Alcohol Advertising Exposure and the Quantity of Alcohol Consumed by Youth.

    Science.gov (United States)

    Naimi, Timothy S; Ross, Craig S; Siegel, Michael B; DeJong, William; Jernigan, David H

    2016-09-01

    Although studies demonstrate that exposure to brand-specific alcohol advertising is associated with an increased likelihood of youth consuming particular brands, the relationship between quantity of brand-specific advertising exposure and quantity of brand-specific consumption has not been firmly established. Using the Alcohol Brand Research Among Underage Drinkers (ABRAND) national sample of 1,031 young drinkers (ages 13-20), this study examined the relationship between their aggregated past-year exposure to advertising (in adstock units, a measure based on gross rating points) for 61 alcohol brands that advertised on the 20 most popular nonsports television programs viewed by underage youth and their aggregated total consumption of those same brands during the past 30 days. Predictive models adjusted for other media exposure, predictors of youth's alcohol consumption, and the consumption of brands not advertised on the 20 shows. For the fully adjusted models, each 100 adstock unit increase in exposure (about 1 SD) was associated with an increase of 5.9 drinks (95% CI [0.9, 11.0 drinks]) consumed during the past 30 days among those with less than 300 units of advertising exposure, and an increase of 55.7 drinks (95% CI [13.9, 97.4 drinks]) among those with 300 or more adstock units of exposure. Among underage youth, the quantity of brand-specific advertising exposure is positively associated with the total quantity of consumption of those advertised brands, even after controlling for the consumption of non-advertised brands. Future research should examine exposure-consumption relationships longitudinally and in other media.

  16. Amount of Televised Alcohol Advertising Exposure and the Quantity of Alcohol Consumed by Youth

    Science.gov (United States)

    Naimi, Timothy S.; Ross, Craig S.; Siegel, Michael B.; DeJong, William; Jernigan, David H.

    2016-01-01

    Objective: Although studies demonstrate that exposure to brand-specific alcohol advertising is associated with an increased likelihood of youth consuming particular brands, the relationship between quantity of brand-specific advertising exposure and quantity of brand-specific consumption has not been firmly established. Method: Using the Alcohol Brand Research Among Underage Drinkers (ABRAND) national sample of 1,031 young drinkers (ages 13–20), this study examined the relationship between their aggregated past-year exposure to advertising (in adstock units, a measure based on gross rating points) for 61 alcohol brands that advertised on the 20 most popular nonsports television programs viewed by underage youth and their aggregated total consumption of those same brands during the past 30 days. Predictive models adjusted for other media exposure, predictors of youth’s alcohol consumption, and the consumption of brands not advertised on the 20 shows. Results: For the fully adjusted models, each 100 adstock unit increase in exposure (about 1 SD) was associated with an increase of 5.9 drinks (95% CI [0.9, 11.0 drinks]) consumed during the past 30 days among those with less than 300 units of advertising exposure, and an increase of 55.7 drinks (95% CI [13.9, 97.4 drinks]) among those with 300 or more adstock units of exposure. Conclusions: Among underage youth, the quantity of brand-specific advertising exposure is positively associated with the total quantity of consumption of those advertised brands, even after controlling for the consumption of non-advertised brands. Future research should examine exposure–consumption relationships longitudinally and in other media. PMID:27588530

  17. Monkey alcohol tissue research resource: banking tissues for alcohol research.

    Science.gov (United States)

    Daunais, James B; Davenport, April T; Helms, Christa M; Gonzales, Steven W; Hemby, Scott E; Friedman, David P; Farro, Jonathan P; Baker, Erich J; Grant, Kathleen A

    2014-07-01

    An estimated 18 million adults in the United States meet the clinical criteria for diagnosis of alcohol abuse or alcoholism, a disorder ranked as the third leading cause of preventable death. In addition to brain pathology, heavy alcohol consumption is comorbid with damage to major organs including heart, lungs, liver, pancreas, and kidneys. Much of what is known about risk for and consequences of heavy consumption derive from rodent or retrospective human studies. The neurobiological effects of chronic intake in rodent studies may not easily translate to humans due to key differences in brain structure and organization between species, including a lack of higher-order cognitive functions, and differences in underlying prefrontal cortical neural structures that characterize the primate brain. Further, rodents do not voluntarily consume large quantities of ethanol (EtOH) and they metabolize it more rapidly than primates. The basis of the Monkey Alcohol Tissue Research Resource (MATRR) is that nonhuman primates, specifically monkeys, show a range of drinking excessive amounts of alcohol (>3.0 g/kg or a 12 drink equivalent per day) over long periods of time (12 to 30 months) with concomitant pathological changes in endocrine, hepatic, and central nervous system (CNS) processes. The patterns and range of alcohol intake that monkeys voluntarily consume parallel what is observed in humans with alcohol use disorders and the longitudinal experimental design spans stages of drinking from the EtOH-naïve state to early exposure through chronic abuse. Age- and sex-matched control animals self-administer an isocaloric solution under identical operant procedures. The MATRR is a unique postmortem tissue bank that provides CNS and peripheral tissues, and associated bioinformatics from monkeys that self-administer EtOH using a standardized experimental paradigm to the broader alcohol research community. This resource provides a translational platform from which we can better

  18. Sequence specific DNA binding by P53 is enhanced by ionizing radiation and is mediated via DNA-PK activity

    International Nuclear Information System (INIS)

    Kachnic, L.A.; Wunsch, H.; Mekeel, K.L.; De Frank, J.S.; Powell, S.N.

    1996-01-01

    Purpose: P53 is known to be involved in the cellular response to DNA damage. It mediates many of its effects by acting as a transcription factor via sequence-specific DNA binding. The half-life of p53 is prolonged following DNA damage, and this results in elevated levels of p53 for a period of 2-8 hours. The increase in p53 is often relatively small, but this produces significant stimulation of a downstream gene such as p21(WAF1/cip1). We investigated post-translational modification of p53 following ionizing radiation damage. Materials and Methods: The response of normal Balb-C mouse fibroblasts (FC) to ionizing radiation (IR, 8 Gy) was measured at 0,3,6,9 and 24 hours, by the levels of p53, p21, flow cytometry and the electrophoretic mobility shift assay (EMSA). EMSA utilized a 26 bp consensus sequence end-labeled oligonucleotide to measure sequence-specific p53 binding. P53 specificity was confirmed by an enhanced mobility shift (retardation) when using p53 antibody. Comparison was made with scid fibroblasts (FS) and FC cells transfected with a plasmid (CX3) containing mutant p53 (alanine-143) or infected with a retrovirus containing the E6 protein of human papilloma virus type 16. Results: The response of p53 to DNA damage shows a 3-fold increase at 3-6 hours, and was not significantly different between FC and FS. FC-CX3 showed detectable basal levels of p53, and a 2-fold further induction of p53 after IR. FC-E6 showed no detectable levels of p53 before or after IR. No induction of p21 or G1/S arrest was seen in FC-CX3 or FC-E6, as has been observed previously. The induction of p21 in FS cells was attenuated and delayed: a 2-3-fold increase seen maximally at 9 hours, compared with a 5-fold increase seen maximally at 3-6 hours in FC cells. The accumulation of cells at the G1/S junction after IR showed the same kinetics as p21 induction: the peak of cells in G1 occurs at 3-6 hours in FC, but not until 9-24 hours in FS. The response is reminiscent of that seen in

  19. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  20. Social and Cultural Contexts of Alcohol Use

    Science.gov (United States)

    Sudhinaraset, May; Wigglesworth, Christina; Takeuchi, David T.

    2016-01-01

    Alcohol use and misuse account for 3.3 million deaths every year, or 6 percent of all deaths worldwide. The harmful effects of alcohol misuse are far reaching and range from individual health risks, morbidity, and mortality to consequences for family, friends, and the larger society. This article reviews a few of the cultural and social influences on alcohol use and places individual alcohol use within the contexts and environments where people live and interact. It includes a discussion of macrolevel factors, such as advertising and marketing, immigration and discrimination factors, and how neighborhoods, families, and peers influence alcohol use. Specifically, the article describes how social and cultural contexts influence alcohol use/misuse and then explores future directions for alcohol research. PMID:27159810

  1. The protective role of low-concentration alcohol in high-fructose induced adverse cardiovascular events in mice.

    Science.gov (United States)

    Wu, Xiaoqi; Pan, Bo; Wang, Ying; Liu, Lingjuan; Huang, Xupei; Tian, Jie

    2018-01-01

    Cardiovascular disease remains a worldwide public health issue. As fructose consumption is dramatically increasing, it has been demonstrated that a fructose-rich intake would increase the risk of cardiovascular disease. In addition, emerging evidences suggest that low concentration alcohol intake may exert a protective effect on cardiovascular system. This study aimed to investigate whether low-concentration alcohol consumption would prevent the adverse effects on cardiovascular events induced by high fructose in mice. From the results of hematoxylin-eosin staining, echocardiography, heart weight/body weight ratio and the expression of hypertrophic marker ANP, we found high-fructose result in myocardial hypertrophy and the low-concentration alcohol consumption would prevent the cardiomyocyte hypertrophy from happening. In addition, we observed low-concentration alcohol consumption could inhibit mitochondria swollen induced by high-fructose. The elevated levels of glucose, triglyceride, total cholesterol in high-fructose group were reduced by low concentration alcohol. Low expression levels of SIRT1 and PPAR-γ induced by high-fructose were significantly elevated when fed with low-concentration alcohol. The histone lysine 9 acetylation (acH3K9) level was decreased in PPAR-γ promoter in high-fructose group but elevated when intake with low concentration alcohol. The binding levels of histone deacetylase SIRT1 were increased in the same region in high-fructose group, while the low concentration alcohol can prevent the increased binding levels. Overall, our study indicates that low-concentration alcohol consumption could inhibit high-fructose related myocardial hypertrophy, cardiac mitochondria damaged and disorders of glucose-lipid metabolism. Furthermore, these findings also provide new insights into histone acetylation-deacetylation mechanisms of low-concentration alcohol treatment that may contribute to the prevention of cardiovascular disease induced by high

  2. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  3. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  4. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 onto plates coated with anti-CD4bs antibodies b12, b6 or the CD4 receptor mimetic, CD4-IgG2...

  5. Alcohol consumption and liver cirrhosis mortality

    DEFF Research Database (Denmark)

    Bentzen, Jan Børsen; Smith, Valdemar

    on the relationship between liver cirrhosis mortality and alcohol consumption is included. The conclusion is that the total level of alcohol consumption as well as the specific beverages - beer, wine and spirits - contributes to liver cirrhosis mortality, but the present study also reveals that directly addressing...

  6. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  7. Alcohol Consumption and Survival after a Breast Cancer Diagnosis

    DEFF Research Database (Denmark)

    Ali, Alaa M G; Schmidt, Marjanka K; Bolla, Manjeet K

    2014-01-01

    BACKGROUND: Evidence for an association of alcohol consumption with prognosis after a diagnosis of breast cancer has been inconsistent. We have reviewed and summarized the published evidence and evaluated the association using individual patient data from multiple case cohorts. METHODS: A MEDLINE...... cancer-specific mortality, with some evidence of a negative association with all-cause mortality. On the basis of a single study, moderate postdiagnosis alcohol intake was associated with a small reduction in breast cancer-specific mortality for women with ER-negative disease. There was no association...... with prediagnosis intake for women with ER-negative disease. CONCLUSION: There was little evidence that pre- or post-diagnosis alcohol consumption is associated with breast cancer-specific mortality for women with ER-positive disease. There was weak evidence that moderate post-diagnosis alcohol intake is associated...

  8. Three amino acid residues bind corn odorants to McinOBP1 in the polyembryonic endoparasitoid of Macrocentrus cingulum Brischke.

    Directory of Open Access Journals (Sweden)

    Tofael Ahmed

    Full Text Available Odorant binding proteins (OBPs play a central role in transporting odorant molecules from the sensillum lymph to olfactory receptors to initiate behavioral responses. In this study, the OBP of Macrocentrus cingulum McinOBP1 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR experiments indicate that the McinOBP1 is expressed mainly in adult antennae, with expression levels differing by sex. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN as a fluorescent probe demonstrated that the McinOBP1 can bind green-leaf volatiles, including aldehydes and terpenoids, but also can bind aliphatic alcohols with good affinity, in the order trans-2-nonenal>cis-3-hexen-1-ol>trans-caryophelle, suggesting a role of McinOBP1 in general odorant chemoreception. We chose those three odorants for further homology modeling and ligand docking based on their binding affinity. The Val58, Leu62 and Glu130 are the key amino acids in the binding pockets that bind with these three odorants. The three mutants, Val58, Leu62 and Glu130, where the valine, leucine and glutamic residues were replaced by alanine, proline and alanine, respectively; showed reduced affinity to these odorants. This information suggests, Val58, Leu62 and Glu130 are involved in the binding of these compounds, possibly through the specific recognition of ligands that forms hydrogen bonds with the ligands functional groups.

  9. Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses.

    Science.gov (United States)

    Goodwin, B J; Moore, J O; Weinberg, J B

    1984-02-01

    Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12-myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5-10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of

  10. Dynamic Exposure to Alcohol Advertising in a Sports Context Influences Implicit Attitudes.

    Science.gov (United States)

    Zerhouni, Oulmann; Bègue, Laurent; Duke, Aaron A; Flaudias, Valentin

    2016-02-01

    Experimental studies investigating the impact of advertising with ecological stimuli on alcohol-related cognition are scarce. This research investigated the cognitive processes involved in learning implicit attitudes toward alcohol after incidental exposure to alcohol advertisements presented in a dynamic context. We hypothesized that incidental exposure to a specific alcohol brand would lead to heightened positive implicit attitudes toward alcohol due to a mere exposure effect. In total, 108 participants were randomly exposed to dynamic sporting events excerpts with and without advertising for a specific brand of alcohol, after completing self-reported measures of alcohol-related expectancies, alcohol consumption, and attitudes toward sport. Participants then completed a lexical decision task and an affective priming task. We showed that participants were faster to detect brand name after being exposed to advertising during a sports game, and that implicit attitudes of participants toward the brand were more positive after they were exposed to advertising, even when alcohol usage patterns were controlled for. Incidental exposure to alcohol sponsorship in sport events impacts implicit attitudes toward the advertised brand and alcohol in general. The effect of incidental advertising on implicit attitudes is also likely to be due to a mere exposure effect. However, further studies should address this point specifically. Copyright © 2016 by the Research Society on Alcoholism.

  11. SERUM IRON PARAMETERS IN ALCOHOLIC CIRRHOSIS, CRYPTOGENIC CIRRHOSIS, CHRONIC HEPATITIS B AND CHRONIC HEPATITIS C

    Directory of Open Access Journals (Sweden)

    Sajeevan K. C

    2016-11-01

    Full Text Available BACKGROUND Regular monitoring of serum iron parameters is helpful for assessing the severity of alcoholic liver disease. Assessment of serum iron parameters are used for screening hereditary haemochromatosis in chronic liver disease. Serum iron parameters in chronic liver disease have not been clearly described in most of the studies. The aim of this study was to assess the serum iron, Total Iron Binding Capacity (TIBC, transferrin saturation and ferritin levels in common chronic liver disease like alcoholic cirrhosis, cryptogenic cirrhosis, chronic hepatitis C and chronic hepatitis B. MATERIALS AND METHODS 110 consecutive patients with chronic liver disease admitted to the Gastroenterology Department, Government Medical College, Kozhikode were selected for the study. The categories of chronic liver disease included in our study were alcoholic cirrhosis (Group I, n = 40, cryptogenic cirrhosis (Group II, n = 30, chronic hepatitis C (Group III, n = 20 and chronic hepatitis B (Group IV, n = 20. Serum iron, ferritin, total iron binding capacity and transferrin saturation were estimated in the fasting sample. Statistical Analysis- Analysis was performed using nonparametric Kruskal-Wallis and Bonferroni test to assess statistical significance of difference of continuous variables among and between groups, respectively. The results were considered statistically significant at the level of p <0.05. RESULTS The serum iron level was normal and total iron binding capacity was low in all the four groups of chronic liver disease. Serum ferritin and transferrin saturation were significantly higher in alcoholic cirrhosis in comparison with cryptogenic cirrhosis and chronic hepatitis B, but was not statistically significant in comparison with chronic hepatitis C. CONCLUSION We observed irregularities in iron status in patients with alcoholic cirrhosis, cryptogenic cirrhosis, chronic hepatitis C and chronic hepatitis B.

  12. Quantitative Molecular Imaging with a Single Gd-Based Contrast Agent Reveals Specific Tumor Binding and Retention in Vivo.

    Science.gov (United States)

    Johansen, Mette L; Gao, Ying; Hutnick, Melanie A; Craig, Sonya E L; Pokorski, Jonathan K; Flask, Chris A; Brady-Kalnay, Susann M

    2017-06-06

    Magnetic resonance imaging (MRI) has become an indispensable tool in the diagnosis and treatment of many diseases, especially cancer. However, the poor sensitivity of MRI relative to other imaging modalities, such as PET, has hindered the development and clinical use of molecular MRI contrast agents that could provide vital diagnostic information by specifically locating a molecular target altered in the disease process. This work describes the specific and sustained in vivo binding and retention of a protein tyrosine phosphatase mu (PTPμ)-targeted, molecular magnetic resonance (MR) contrast agent with a single gadolinium (Gd) chelate using a quantitative MRI T 1 mapping technique in glioma xenografts. Quantitative T 1 mapping is an imaging method used to measure the longitudinal relaxation time, the T 1 relaxation time, of protons in a magnetic field after excitation by a radiofrequency pulse. T 1 relaxation times can in turn be used to calculate the concentration of a gadolinium-containing contrast agent in a region of interest, thereby allowing the retention or clearance of an agent to be quantified. In this context, retention is a measure of molecular contrast agent binding. Using conventional peptide chemistry, a PTPμ-targeted peptide was linked to a chelator that had been conjugated to a lysine residue. Following complexation with Gd, this PTPμ-targeted molecular contrast agent containing a single Gd ion showed significant tumor enhancement and a sustained increase in Gd concentration in both heterotopic and orthotopic tumors using dynamic quantitative MRI. This single Gd-containing PTPμ agent was more effective than our previous version with three Gd ions. Differences between nonspecific and specific agents, due to specific tumor binding, can be determined within the first 30 min after agent administration by examining clearance rates. This more facile chemistry, when combined with quantitative MR techniques, allows for widespread adoption by academic

  13. A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

    Science.gov (United States)

    Kim, Seonghun

    2018-02-01

    Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats.

    Science.gov (United States)

    Grissom, Elin M; Hawley, Wayne R; Hodges, Kelly S; Fawcett-Patel, Jessica M; Dohanich, Gary P

    2013-04-01

    According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males

  15. 41 CFR 60-300.24 - Drugs and alcohol.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Drugs and alcohol. 60-300... VETERANS Discrimination Prohibited § 60-300.24 Drugs and alcohol. (a) Specific activities permitted. The contractor: (1) May prohibit the illegal use of drugs and the use of alcohol at the workplace by all...

  16. 41 CFR 60-741.24 - Drugs and alcohol.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Drugs and alcohol. 60-741... INDIVIDUALS WITH DISABILITIES Discrimination Prohibited § 60-741.24 Drugs and alcohol. (a) Specific activities permitted. The contractor: (1) May prohibit the illegal use of drugs and the use of alcohol at the workplace...

  17. 41 CFR 60-250.24 - Drugs and alcohol.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Drugs and alcohol. 60-250... VETERANS Discrimination Prohibited § 60-250.24 Drugs and alcohol. (a) Specific activities permitted. The contractor: (1) May prohibit the illegal use of drugs and the use of alcohol at the workplace by all...

  18. The relationship between prenatal care, personal alcohol abuse and alcohol abuse in the home environment

    Science.gov (United States)

    GREKIN, EMILY R.; ONDERSMA, STEVEN J.

    2013-01-01

    Aims Nearly one-fourth of African-American women receive no prenatal care during the first trimester of pregnancy. The aim of the current study is to identify factors that underlie inadequate prenatal care among African-American women. Maternal alcohol abuse has been examined as one risk factor for inadequate prenatal care, but findings have been inconsistent, perhaps because (a) alcohol use during pregnancy is substantially under-reported and (b) studies have not considered the wider social network in which maternal alcohol use takes place. The current study attempts to clarify relationships between personal alcohol use, alcohol use in the home environment, and prenatal care in a sample of post-partum women. Methods Participants were 107 low-income, primarily African-American women. All participants completed a computer-based screening which assessed personal and environmental alcohol use, prenatal care and mental health. Findings Environmental alcohol use was related to delayed prenatal care while personal alcohol use was not. More specifically, after controlling for demographic variables, the presence of more than three person-episodes of binge drinking in a woman’s home environment increased the odds of seriously compromized prenatal care by a factor of seven. Conclusions Findings suggest the need to further assess environmental alcohol use and to examine the reliability of personal alcohol use measures. PMID:24391354

  19. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

    Science.gov (United States)

    An, Bo; DesRochers, Teresa M; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David L

    2013-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    International Nuclear Information System (INIS)

    Hayakawa, Hiroshi; Fujikane, Aya; Ito, Riyoko; Matsumoto, Masaki; Nakayama, Keiichi I.; Sekiguchi, Mutsuo

    2010-01-01

    Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H 2 O 2 . → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H 2 O 2 . -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

  1. mRNA Display Selection of a High-Affinity, Modification-Specific Phospho-IκBα-Binding Fibronectin

    Science.gov (United States)

    Olson, C. Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W.

    2009-01-01

    The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IκBα. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IκBα peptide with Kd = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IκBα from mammalian cell extract and stabilizes phospho-IκBα in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IκB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors. PMID:18590330

  2. mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin.

    Science.gov (United States)

    Olson, C Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W

    2008-08-15

    The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IkappaBalpha. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IkappaBalpha peptide with K d = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IkappaBalpha from mammalian cell extract and stabilizes phospho-IkappaBalpha in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IkappaB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.

  3. Specific in vivo binding in the rat brain of [18F]RP 62203: A selective 5-HT2A receptor radioligand for positron emission tomography

    International Nuclear Information System (INIS)

    Besret, Laurent; Dauphin, Francois; Huard, Cecile; Lasne, Marie-Claire; Vivet, Richard; Mickala, Patrick; Barbelivien, Alexandra; Baron, Jean-Claude

    1996-01-01

    In vivo pharmacokinetic and brain binding characteristics of [ 18 F]RP 62203, a selective high-affinity serotonergic 5-HT 2A receptor antagonist, were assessed in the rat following intravenous injection of trace amount of the radioligand. The radioactive distribution profile observed in the brain 60 min after injection was characterized by greater than fourfold higher uptake in neocortex as compared to cerebellum (0.38 ± 0.07% injected dose/g, % ID/g and 0.08 ± 0.01 ID/g, respectively), consistent with in vivo specific binding to the 5-HT 2A receptor. Furthermore, specific [ 18 F]RP 62203 binding significantly correlated with the reported in vitro distribution of 5-HT 2A receptors, but not with known concentration profiles of dopaminergic D 2 or adrenergic α 1 receptors. Finally, detectable specific binding was abolished by pretreatment with large doses of ritanserin, a selective 5-HT 2A antagonist, which resulted in uniform uptakes across cortical, striatal and cerebellar tissues. Thus, [ 18 F]RP 62203 appears to be a promising selective tool to visualize and quantify 5-HT 2A brain receptors in vivo with positron emission tomography

  4. Specificity and autoregulation of Notch binding by tandem WW domains in suppressor of Deltex.

    Science.gov (United States)

    Jennings, Martin D; Blankley, Richard T; Baron, Martin; Golovanov, Alexander P; Avis, Johanna M

    2007-09-28

    WW domains target proline-t