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Sample records for affected sib-pair asp

  1. LOD wars: The affected-sib-pair paradigm strikes back!

    SciTech Connect

    Farrall, M.

    1997-03-01

    In a recent letter, Greenberg et al. aired their concerns that the affected-sib-pair (ASP) approach was becoming excessively popular, owing to misconceptions and ignorance of the properties and limitations of both the ASP and the classic LOD-score approaches. As an enthusiast of using the ASP approach to map susceptibility genes for multifactorial traits, I would like to contribute a few comments and explanatory notes in defense of the ASP paradigm. 18 refs.

  2. Mathematical Assumptions versus Biological Reality: Myths in Affected Sib Pair Linkage Analysis

    PubMed Central

    Elston, Robert C.; Song, Danhong; Iyengar, Sudha K.

    2005-01-01

    Affected sib pair (ASP) analysis has become common ever since it was shown that, under very specific assumptions, ASPs afford a powerful design for linkage analysis. In 2003, Vieland and Huang, on the basis of a “fundamental heterogeneity equation,” proved that heterogeneity and epistasis are confounded in ASP linkage analysis. A much more serious limitation of ASP linkage analysis is the implicit assumption that randomly sampled sib pairs share half their alleles identical by descent at any locus, whereas a critical assumption underlying Vieland and Huang’s proof is that of joint Hardy-Weinberg equilibrium proportions at two trait loci. These are considered as examples of mathematical assumptions that may not always reflect biological reality. More-robust sib-pair designs and appropriate methods for their analysis have long been available. PMID:15540158

  3. Reply to Farrall. LOD wars: The affected-sib-pair paradigm strikes back!

    SciTech Connect

    Greenberg, D.A.; Hodge, S.E.; Vieland, V.J.; Spence, M.A.

    1997-03-01

    Farrall, in his comments about our previous letter, eloquently points out some of the advantages of affected-sib-pair (ASP) analysis. It may surprise him to learn that we do not disagree with most of his comments, but we do feel that a few of them could be potentially misleading. Farrall says that {open_quotes}ASP tests are nonparametric{close_quote} in the limited sense that the investigator does not have to declare (or have prior knowledge of) an explicit set of genetic parameters before undertaking a meaningful analysis. He could have added that one does not have the option of changing genetic parameters. One does not have to declare genetic parameters, because the design of the test is such that certain assumptions are inherent in the method. These assumptions are not obvious - and even may not be known - in the nonparametric methods. However, they are still there. 4 refs.

  4. Affected-sib-pair analyses reveal support of prior evidence for a susceptibility locus for bipolar disorder, on 21q

    SciTech Connect

    Detera-Wadleigh, S.D.; Badner, J.A.; Goldin, L.R.

    1996-06-01

    In 22 multiplex pedigrees screened for linkage to bipolar disorder, by use of 18 markers on chromosome 21q, single-locus affected-sib-pair (ASP) analysis detected a high proportion (57%-62%) of alleles shared identical by descent (IBD), with P values of .049-.0008 on nine marker loci. Multilocus ASP analyses revealed locus trios in the distal region between D21S270 and D21S171, with excess allele sharing (nominal P values <.01) under two affection-status models, ASM I (bipolars and schizoaffectives) and ASM II (ASM I plus recurrent unipolars). In addition, under ASM I, the proximal interval spanned by D21S1436 and D21S65 showed locus trios with excess allele sharing (nominal P values of .03-.0003). These findings support prior evidence that a susceptibility locus for bipolar disorder is on 21q. 38 refs., 4 tabs.

  5. Affected sib-pair interval mapping and exclusion for complex genetic traits: Inferring identity by descent status from relatives

    SciTech Connect

    Hauser, E.R.; Boehnke, M.; Guo, S.W.

    1994-09-01

    Affected sib-pair (ASP) methods provide a useful approach for the initial genetic mapping of complex diseases for which mode of inheritance is uncertain. Risch described a method for interval mapping and exclusion based on the ratio lambda comparing disease risk in the first degree relatives of affected individuals to disease risk in the general population. He assumed marker identity by descent (IBD) status for the ASP could be deduced from parental genotypes. For late onset diseases such as type 2 diabetes, parents may be dead or otherwise unavailable, so that marker IBD status generally cannot be inferred with certainty. Guo has developed efficient methods for probabilistic determination of marker IBD sharing for two or more loci. We have combined and extended the methods of Risch and Guo to carry out interval mapping and exclusion when parents are missing but other relatives such as additional siblings are available. Our method is based on calculating the likelihood of marker data of the ASP and their relatives conditional on the disease status of the ASP, as a function of lambda and the position of the disease locus within the genetic map. We currently are using this method to compare the information to detect or exclude linkage provided by various types of ASP nuclear families -- zero, one, or two typed parents and zero, one, two, or more additional siblings -- as a function of sample size, marker density and informativity, and risk ratio lambda.

  6. Impact of parental relationships in maximum lod score affected sib-pair method.

    PubMed

    Leutenegger, Anne-Louise; Génin, Emmanuelle; Thompson, Elizabeth A; Clerget-Darpoux, Françoise

    2002-11-01

    Many studies are done in small isolated populations and populations where marriages between relatives are encouraged. In this paper, we point out some problems with applying the maximum lod score (MLS) method (Risch, [1990] Am. J. Hum. Genet. 46:242-253) in these populations where relationships exist between the two parents of the affected sib-pairs. Characterizing the parental relationships by the kinship coefficient between the parents (f), the maternal inbreeding coefficient (alpha(m), and the paternal inbreeding coefficient (alpha(p)), we explored the relationship between the identity by descent (IBD) vector expected under the null hypothesis of no linkage and these quantities. We find that the expected IBD vector is no longer (0.25, 0.5, 0.25) when f, alpha(m), and alpha(p) differ from zero. In addition, the expected IBD vector does not always follow the triangle constraints recommended by Holmans ([1993] Am. J. Hum. Genet. 52:362-374). So the classically used MLS statistic needs to be adapted to the presence of parental relationships. We modified the software GENEHUNTER (Kruglyak et al. [1996] Am. J. Hum. Genet. 58: 1347-1363) to do so. Indeed, the current version of the software does not compute the likelihood properly under the null hypothesis. We studied the adapted statistic by simulating data on three different family structures: (1) parents are double first cousins (f=0.125, alpha(m)=alpha(p)=0), (2) each parent is the offspring of first cousins (f=0, alpha(m)=alpha(p)=0.0625), and (3) parents are related as in the pedigree from Goddard et al. ([1996] Am. J. Hum. Genet. 58:1286-1302) (f=0.109, alpha(m)=alpha(p)=0.0625). The appropriate threshold needs to be derived for each case in order to get the correct type I error. And using the classical statistic in the presence of both parental kinship and parental inbreeding almost always leads to false conclusions.

  7. A combined analysis of D22S278 marker alleles in affected sib-pairs: Support for a susceptibility locus for schizophrenia at chromosome 22q12

    SciTech Connect

    Gill, M.; Vallada, H.; Collier, D.

    1996-02-16

    Several groups have reported weak evidence for linkage between schizophrenia and genetic markers located on chromosome 22q using the lod score method of analysis. However these findings involved different genetic markers and methods of analysis, and so were not directly comparable. To resolve this issue we have performed a combined analysis of genotypic data from the marker D22S278 in multiply affected schizophrenic families derived from 11 independent research groups worldwide. This marker was chosen because it showed maximum evidence for linkage in three independent datasets. Using the affected sib-pair method as implemented by the program ESPA, the combined dataset showed 252 alleles shared compared with 188 alleles not shared (chi-square 9.31, 1df, P = 0.001) where parental genotype data was completely known. When sib-pairs for whom parental data was assigned according to probability were included the number of alleles shared was 514.1 compared with 437.8 not shared (chi-square 6.12, 1df, P = 0.006). Similar results were obtained when a likelihood ratio method for sib-pair analysis was used. These results indicate that there may be a susceptibility locus for schizophrenia at 22q12. 27 refs., 3 tabs.

  8. Transmission-ratio distortion and allele sharing in affected sib pairs: a new linkage statistic with reduced bias, with application to chromosome 6q25.3.

    PubMed

    Lemire, Mathieu; Roslin, Nicole M; Laprise, Catherine; Hudson, Thomas J; Morgan, Kenneth

    2004-10-01

    We studied the effect of transmission-ratio distortion (TRD) on tests of linkage based on allele sharing in affected sib pairs. We developed and implemented a discrete-trait allele-sharing test statistic, Sad, analogous to the Spairs test statistic of Whittemore and Halpern, that evaluates an excess sharing of alleles at autosomal loci in pairs of affected siblings, as well as a lack of sharing in phenotypically discordant relative pairs, where available. Under the null hypothesis of no linkage, nuclear families with at least two affected siblings and one unaffected sibling have a contribution to Sad that is unbiased, with respect to the effects of TRD independent of the disease under study. If more distantly related unaffected individuals are studied, the bias of Sad is generally reduced compared with that of Spairs, but not completely. Moreover, Sad has higher power, in some circumstances, because of the availability of unaffected relatives, who are ignored in affected-only analyses. We discuss situations in which it may be an efficient use of resources to genotype unaffected relatives, which would give insights for promising study designs. The method is applied to a sample of pedigrees ascertained for asthma in a chromosomal region in which TRD has been reported. Results are consistent with the presence of transmission distortion in that region. PMID:15322985

  9. OB gene not linked to human obesity in Mexican American affected sib pairs from Starr County, Texas.

    PubMed

    Bray, M S; Boerwinkle, E; Hanis, C L

    1996-11-01

    Obesity is a highly prevalent disease, which is associated with a number of chronic conditions and, as such, represents a major public health burden. Numerous studies indicate that there is a genetic component contributing to interindividual variability in obesity. The discovery of the ob gene in mice, mutations in which produce extreme obesity and non-insulin-dependent diabetes mellitus (NIDDM), provides a prime candidate gene for human obesity. We investigated linkage between the human OB gene and obesity in a sample of Mexican Americans from Starr County, Texas. Markers D7S635 and D7S1875, estimated to lie within a region approximately 290 to 400 kb proximal to the OB gene, were used to genotype 177 obese individuals distributed in 64 sibships. Obesity was defined as a body mass index (BMI) above 30 kg/m2. Linkage analyses for affected sibling pairs provided no evidence for linkage in this sample. In addition, differences between siblings for weight, BMI, systolic and diastolic blood pressure, percent body fat, waist-to-hip ratio, and blood lipid measures were not significantly related to number of alleles shared identical by state (IBS) for either of the two markers. While the OB gene may be involved in the metabolic sequences leading to obesity, the present linkage results do not support the existence of common genetic variation at or near the OB locus that increases risk for human obesity. PMID:8882881

  10. Association of ADH and ALDH Genes With Alcohol Dependence in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) Sample

    PubMed Central

    Kuo, Po-Hsiu; Kalsi, Gursharan; Prescott, Carol A.; Hodgkinson, Colin A.; Goldman, David; van den Oord, Edwin J.; Alexander, Jeffry; Jiang, Cizhong; Sullivan, Patrick F.; Patterson, Diana G.; Walsh, Dermot; Kendler, Kenneth S.; Riley, Brien P.

    2008-01-01

    Background: The genes coding for ethanol metabolism enzymes [alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] have been widely studied for their influence on the risk to develop alcohol dependence (AD). However, the relation between polymorphisms of these metabolism genes and AD in Caucasian subjects has not been clearly established. The present study examined evidence for the association of alcohol metabolism genes with AD in the Irish Affected Sib Pair Study of alcohol dependence. Methods: We conducted a case–control association study with 575 independent subjects who met Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, AD diagnosis and 530 controls. A total of 77 single nucleotide polymorphisms (SNPs) in the seven ADH (ADH1-7) and two ALDH genes (ALDH1A1 and ALDH2) were genotyped using the Illumina GoldenGate protocols. Several statistical procedures were implemented to control for false discoveries. Results: All markers with minor allele frequency greater than 0.01 were in Hardy–Weinberg equilibrium. Numerous SNPs in ADH genes showed association with AD, including one marker in the coding region of ADH1C (rs1693482 in exon6, Ile271Gln). Haplotypic association was observed in the ADH5 and ADH1C genes, and in a long haplotype block formed by the ADH1A and ADH1B loci. We detected two significant interactions between pairs of markers in intron 6 of ADH6 and intron 12 of ALDH2 (p = 5 × 10−5), and 5′ of both ADH4 and ADH1A (p = 2 × 10−4). Conclusion: We found evidence for the association of several ADH genes with AD in a sample of Western European origin. The significant interaction effects between markers in ADH and ALDH genes suggest possible epistatic roles between alcohol metabolic enzymes in the risk for AD. PMID:18331377

  11. Complete multipoint sib-pair analysis of qualitative and quantitative traits

    SciTech Connect

    Kruglyak, L.; Lander, E.S.

    1995-08-01

    Sib-pair analysis is an increasingly important tool for genetic dissection of complex traits. Current methods for sib-pair analysis are primarily based on studying individual genetic markers one at a time and thus fail to use the full inheritance information provided by multipoint linkage analysis. In this paper, we describe how to extract the complete multipoint inheritance information for each sib pair. We then describe methods that use this information to map loci affecting traits, thereby providing a unified approach to both qualitative and quantitative traits. Specifically, complete multipoint approaches are presented for (1) exclusion mapping of qualitative traits; (2) maximum-likelihood mapping of qualitative traits; (3) information-content mapping, showing the extent to which all inheritance information has been extracted at each location in the genome; and (4) quantitative-trait mapping, by two parametric methods and one nonparametric method. In addition, we explore the effects of marker density, marker polymorphism, and availability of parents on the information content of a study. We have implemented the analysis methods in a new computer package, MAPMAKER/SIBS. With this computer package, complete multipoint analysis with dozens of markers in hundreds of sib pairs can be carried out in minutes. 25 refs., 8 figs.

  12. The phenotypic difference discards sib-pair QTL linkage information

    SciTech Connect

    Wright, F.A. |

    1997-03-01

    Kruglyak and Lander provide an important synthesis of methods for (IBD) sib-pair linkage mapping, with an emphasis on the use of complete multipoint inheritance information for each sib pair. These procedures are implemented in the computer program MAPMAKER/SIBS, which performs interval mapping for dichotomous and quantitative traits. The authors present three methods for mapping quantitative trait loci (QTLs): a variant of the commonly used Haseman-Elston regression approach, a maximum-likelihood procedure involving variance components, and a rank-based nonparametric procedure. These approaches and related work use the magnitude of the difference in the sibling phenotype values for each sib pair as the observation for analysis. Linkage is detected if siblings sharing more alleles IBD have similar phenotypes (i.e., a small difference in the phenotype values), while siblings sharing fewer alleles IBD have less similar phenotypes. Such techniques have been used to detect linkage for a number of quantitative traits. However, the exclusive reliance on the phenotypic differences may be due in large part to historical inertia. A likelihood argument is presented here to show that, under certain classical assumptions, the phenotypic differences do not contain the full likelihood information for QTL mapping. Furthermore, considerable gains in power to detect linkage can be achieved with an expanded likelihood model. The development here is related to previous work, which incorporates the full set of phenotypic data using likelihood and robust quasi-likelihood methods. The purpose of this letter is not to endorse a particular approach but to spur research in alternative and perhaps more powerful linkage tests. 17 refs.

  13. Consanguinity and the sib-pair method: An approach using identity by descent between and within individuals

    SciTech Connect

    Genin, E.; Clerget-Darpoux, F.

    1996-11-01

    To test for linkage between a trait and a marker, one can consider identical marker alleles in related individuals, for instance, sibs. For recessive diseases, it has been shown that some information may be gained from the identity by descent (IBD) of the two alleles of an affected inbred individual at the marker locus. The aim of this paper is to extend the sib-pair method of linkage analysis to the situation of sib pairs sampled from consanguineous populations. This extension takes maximum advantage of the information provided by both the IBD pattern between sibs and allelic identity within each sib of the pair. This is possible through the use of the condensed identity coefficients. Here, we propose a new test of linkage based on a {Chi}{sup 2}. We compare the performance of this test with that of the classical {Chi}{sup 2} test based on the distribution of sib pairs sharing 0, 1, or 2 alleles IBD. For sib pairs from first-cousin matings, the proposed test can better detect the role of a disease-susceptibility (DS) locus. Its power is shown to be greater than that of the classical test, especially for models where the DS allele may be common and incompletely penetrant; that is to say for situations that may be encountered in multifactorial diseases. A study of the impact of inbreeding on the expected proportions of sib pairs sharing 0, 1, or 2 alleles IBD is also performed here. Ignoring inbreeding, when in fact inbreeding exists, increases the rate of type I errors in tests of linkage. 21 refs., 8 figs., 9 tabs.

  14. An extreme-sib-pair genome scan for genes regulating blood pressure.

    PubMed

    Xu, X; Rogus, J J; Terwedow, H A; Yang, J; Wang, Z; Chen, C; Niu, T; Wang, B; Xu, H; Weiss, S; Schork, N J; Fang, Z

    1999-06-01

    Hypertension, a risk factor for many cardiovascular, cerebrovascular, and renal diseases, affects one in four Americans, at an annual cost of>$30 billion. Although genetic mutations have been identified in rare forms of hypertension, including Liddle syndrome and glucocorticoid-remediable aldosteronism, the abundance of plausible candidate genes and potential environmental risk factors has complicated the genetic dissection of more prevalent essential hypertension. To search systematically for chromosomal regions containing genes that regulate blood pressure, we scanned the entire autosomal genome by using 367 polymorphic markers. Our study population, selected from a blood-pressure screen of >200,000 Chinese adults, comprises rare but highly efficient extreme sib pairs (207 discordant, 258 high concordant, and 99 low concordant) and all but a single parent of these sibs. By virtue of the sampling design, the number of sib pairs, and the availability of genotyped parents, this study represents one of the most powerful of its kind. Although no regions achieved a 5% genomewide significance level, maximum LOD-score values were >2.0 (unadjusted P<.001) for regions containing five markers (D3S2387, D11S2019, D15S657, D16S3396, and D17S1303), in our primary analysis. Other promising regions identified through secondary analyses include loci near D4S3248, D7S2195, D10S1423, D20S470, D20S482, D21S2052, PAH, and AGT.

  15. Neuropsychological and dimensional behavioral trait profiles in Costa Rican ADHD sib pairs: Potential intermediate phenotypes for genetic studies.

    PubMed

    Peskin, Viviana A; Ordóñez, Anna; Mackin, R Scott; Delucchi, Kevin; Monge, Silvia; McGough, James J; Chavira, Denise A; Berrocal, Monica; Cheung, Erika; Fournier, Eduardo; Badner, Judith A; Herrera, Luis Diego; Mathews, Carol A

    2015-06-01

    Attention deficit hyperactivity disorder (ADHD) is associated with substantial functional impairment in children and in adults. Many individuals with ADHD have clear neurocognitive deficits, including problems with visual attention, processing speed, and set shifting. ADHD is etiologically complex, and although genetic factors play a role in its development, much of the genetic contribution to ADHD remains unidentified. We conducted clinical and neuropsychological assessments of 294 individuals (269 with ADHD) from 163 families (48 multigenerational families created using genealogical reconstruction, 78 affected sib pair families, and 37 trios) from the Central Valley of Costa Rica (CVCR). We used principal components analysis (PCA) to group neurocognitive and behavioral variables using the subscales of the Child Behavior Checklist (CBCL) and 15 neuropsychological measures, and created quantitative traits for heritability analyses. We identified seven cognitive and two behavioral domains. Individuals with ADHD were significantly more impaired than their unaffected siblings on most behavioral and cognitive domains. The verbal IQ domain had the highest heritability (92%), followed by auditory attention (87%), visual processing speed and problem solving (85%), and externalizing symptoms (81%). The quantitative traits identified here have high heritabilities, similar to the reported heritability of ADHD (70-90%), and may represent appropriate alternative phenotypes for genetic studies. The use of multigenerational families from a genetically isolated population may facilitate the identification of ADHD risk genes in the face of phenotypic and genetic heterogeneity. PMID:25832558

  16. Neuropsychological and dimensional behavioral trait profiles in Costa Rican ADHD sib pairs: Potential intermediate phenotypes for genetic studies.

    PubMed

    Peskin, Viviana A; Ordóñez, Anna; Mackin, R Scott; Delucchi, Kevin; Monge, Silvia; McGough, James J; Chavira, Denise A; Berrocal, Monica; Cheung, Erika; Fournier, Eduardo; Badner, Judith A; Herrera, Luis Diego; Mathews, Carol A

    2015-06-01

    Attention deficit hyperactivity disorder (ADHD) is associated with substantial functional impairment in children and in adults. Many individuals with ADHD have clear neurocognitive deficits, including problems with visual attention, processing speed, and set shifting. ADHD is etiologically complex, and although genetic factors play a role in its development, much of the genetic contribution to ADHD remains unidentified. We conducted clinical and neuropsychological assessments of 294 individuals (269 with ADHD) from 163 families (48 multigenerational families created using genealogical reconstruction, 78 affected sib pair families, and 37 trios) from the Central Valley of Costa Rica (CVCR). We used principal components analysis (PCA) to group neurocognitive and behavioral variables using the subscales of the Child Behavior Checklist (CBCL) and 15 neuropsychological measures, and created quantitative traits for heritability analyses. We identified seven cognitive and two behavioral domains. Individuals with ADHD were significantly more impaired than their unaffected siblings on most behavioral and cognitive domains. The verbal IQ domain had the highest heritability (92%), followed by auditory attention (87%), visual processing speed and problem solving (85%), and externalizing symptoms (81%). The quantitative traits identified here have high heritabilities, similar to the reported heritability of ADHD (70-90%), and may represent appropriate alternative phenotypes for genetic studies. The use of multigenerational families from a genetically isolated population may facilitate the identification of ADHD risk genes in the face of phenotypic and genetic heterogeneity.

  17. Path analytic, sib-pair linkage and co-twin control studies of asthma and atopy

    SciTech Connect

    Duffy, D.L.; Healey, S.C.; Martin, N.G.

    1994-09-01

    Asthma and atopy are complex traits with multifactorial determinants, and require appropriate choice of phenotypes and analyses, including a linkage analysis of the putative 11q atopy locus. Participants in a large registry-based twin study of asthma were invited to take part in clinical testing. A total of 863 individuals including 419 complete twin pairs (where one or both members reported a history of wheeze) underwent histamine inhalation challenge, allergen skin prick testing, and venesection. Total serum immunoglobulin E (IgE) and bronchial responsiveness (BR) to histamine were highest in those who had wheezed most recently, and whose skin tests demonstrated allergy to house dust mite, cockroach, and rye grass. In ascertainment-corrected path analyses (FISHER), the heritability of IgE and BR were both 60%. Monozygotic (MZ) co-twin control analyses suggested house dust mite sensitization was the single strongest environmentally controlled risk factor for wheeze, while path analyses suggested genetic determination. In dizygotic (DZ) co-twin control analyses, sensitization to grasses was also an important predictor, suggesting pollinosis to be genetically correlated with wheezing, rather than causative. Multivariate path analyses suggested separate (correlated) genetic factors for BR, IgE, and allergy to house dust mite. A sib-pair (Haseman-Elston) linkage analysis of 220 DZ twin pairs did not support linkage to the high-affinity IgE receptor beta-subunit gene on 11q13 of atopy or BR. More recent linkage analyses that include parental genotyping will also be discussed. We conclude that the atopic phenotype consists of a number of traits with specific genetic allergens. Exposure to particular allergens can then cause specific outcomes, such as asthma.

  18. A genome-wide search for linkage to allergic rhinitis in Danish sib-pair families.

    PubMed

    Kruse, Lisbeth Venø; Nyegaard, Mette; Christensen, Ulla; Møller-Larsen, Steffen; Haagerup, Annette; Deleuran, Mette; Hansen, Lars Gudmund; Venø, Stine Krogh; Goossens, Dirk; Del-Favero, Jurgen; Børglum, Anders Dupont

    2012-09-01

    Allergic rhinitis (AR) is a complex disorder with a polygenic, multifactorial aetiology. Twin studies have found the genetic contribution to be substantial. We collected and clinically characterised a sample consisting of 127 Danish nuclear families with at least two siblings suffering from AR or allergic conjunctivitis including 540 individuals (286 children and 254 parents). A whole-genome linkage scan, using 424 microsatellite markers, was performed on both this sample and an earlier collected sample consisting of 130 families with atopic dermatitis and other atopic disorders. A third sib-pair family sample, which was previously collected and genotyped, was added to the analysis increasing the total sample size to 357 families consisting of 1508 individuals. In total, 190 families with AR was included. The linkage analysis software Genehunter NPL, Genehunter MOD, and Genehunter Imprinting were used to obtain nonparametric and parametric linkage results. Family-based association analysis of positional candidate SNPs was carried out using the FBAT program. We obtained genome-wide significant linkage to a novel AR locus at 1p13 and suggestive linkage to two novel regions at 1q31-q32 and 20p12, respectively. Family-based association analysis of SNPs in the candidate locus DNND1B/CRB1 at 1q31 showed no significant association and could not explain the linkage signal observed. Suggestive evidence of linkage was also obtained at three AR loci previously reported (2q14-q23, 2q23, and 12p13) and indication of linkage was observed at a number of additional loci. Likely maternal imprinting was observed at 2q23, and possible maternal imprinting at 3q28. PMID:22419170

  19. Optimal selection of sib pairs from random samples for linkage analysis of a QTL using the EDAC test.

    PubMed

    Dolan, C V; Boomsma, D I

    1998-05-01

    Percentages of extremely concordant and extremely discordant sib pairs are calculated that maximize the power to detect a quantitative trait locus (QTL) under a variety of circumstances using the EDAC test. We assume a large fixed number of randomly sampled sib pairs, such as one would hope to find in the large twin registries, and limited resources to genotype a certain number of selected sib pairs. Our aim is to investigate whether optimal selection can be achieved when prior knowledge concerning the QTL gene action, QTL allele frequency, QTL effect size, and background (residual) sib correlation is limited or absent. To this end we calculate the best selection percentages for a large number of models, which differ in QTL gene action allele frequency, background correlation, and QTL effect size. By averaging these percentages over gene action, over allele frequency, over gene action, and over allele frequencies, we arrive at general recommendations concerning selection percentages. The soundness of these recommendations is subsequently in a number of test cases. PMID:9670595

  20. Affecteds-only linkage methods are not a panacea

    SciTech Connect

    Greenberg, D.A.; Hodge, S.E. |; Vieland, V.J.; Spence, M.A.

    1996-04-01

    Affected-sib-pair (ASP) methods and their variations, such as the affected-pedigree-member (APM) method, have become preferred methods of analysis. Three reasons are usually given as the advantages of ASP/APM methods over LOD-score analysis (likelihood-maximization techniques). First, the most frequently cited is that ASP/APM analysis is non-parametric, whereas LOD-score analysis requires specification of a genetic model. A related consideration is that penetrance is not a confounding factor in ASP/APM analysis, as it is with LOD scores. Second, ASP/APM methods require the collection of data from only a limited number of family members - and, presumably, from those most motivated to help research in the disease, i.e., those affected. A third reason concerns the ease and intuitive appeal of the theory and calculations. Sib-pair analysis is easily understood and can be done on the {open_quotes}back of the envelope.{close_quotes} Except for the more sophisticated ASP methods, one need only count the numbers of sibs sharing no, one, or two alleles in common and apply conventional statistics, to get an answer (although APM methods do require more sophisticated analysis). LOD-score analysis, on the other hand, appears more complex, requiring likelihood maximization, computers and sophisticated programs, and the assumption of a mode of inheritance. 32 refs.

  1. A nonparametric regression-based linkage scan of rheumatoid factor-IgM using sib-pair squared sums and differences

    PubMed Central

    Ghosh, Saurabh; Rao, P Samba Siva; De, Gourab; Majumder, Partha P

    2007-01-01

    Parametric linkage methods for quantitative trait locus mapping require explicit specification of the probability model of the quantitative trait and hence can lead to misleading linkage inferences when the model assumptions are not valid. Ghosh and Majumder developed a nonparametric regression method based on kernel-smoothing for linkage mapping of quantitative trait locus using squared differences in trait values of independent sib pairs, which is relatively more robust than parametric methods with respect to violations in distributional assumptions. In this study, we modify the above mentioned nonparametric regression method by considering local linear polynomials instead of the Nadaraya-Watson estimator and squared sums of sib-pair trait values in addition to squared differences to perform a genome-wide scan of rheumatoid factor-IgM levels on sib pairs in the Genetic Analysis Workshop 15 simulated data set. We obtain significant evidence of linkage very close to the quantitative trait locus controlling for RF-IgM. We find that the simultaneous use of squared differences and squared sums increases the power to detect linkage compared to using only squared differences. However, because of all the sib pairs are selected for rheumatoid arthritis, there is reduced variance of RF-IgM values, and empirical power to detect linkage is not very high. We also compare the performance of our method with two linear regression approaches: the classical Haseman-Elston method using squared sib-pair trait differences and its extension proposed by Elston et al. using mean-corrected sib-pair cross-products. We find that the proposed nonparametric method yields more power than the linear regression approaches. PMID:18466603

  2. A genome-wide sib-pair scan for quantitative language traits reveals linkage to chromosomes 10 and 13

    PubMed Central

    Evans, P. D.; Mueller, K. L.; Gamazon, E. R.; Cox, N. J.; Tomblin, J. B.

    2016-01-01

    Although there is considerable evidence that individual differences in language development are highly heritable, there have been few genome-wide scans to locate genes associated with the trait. Previous analyses of language impairment have yielded replicable evidence for linkage to regions on chromosomes 16q, 19q, 13q (within lab) and at 13q (between labs). Here we report the first linkage study to screen the continuum of language ability, from normal to disordered, as found in the general population. 383 children from 147 sib-ships (214 sib-pairs) were genotyped on the Illumina® Linkage IVb Marker Panel using three composite language-related phenotypes and a measure of phonological memory (PM). Two regions (10q23.33; 13q33.3) yielded genome-wide significant peaks for linkage with PM. A peak suggestive of linkage was also found at 17q12 for the overall language composite. This study presents two novel genetic loci for the study of language development and disorders, but fails to replicate findings by previous groups. Possible reasons for this are discussed. PMID:25997078

  3. Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

    PubMed Central

    Batista, F R; Hernández, L; Fernández, J R; Arrieta, J; Menéndez, C; Gómez, R; Támbara, Y; Pons, T

    1999-01-01

    beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases. PMID:9895294

  4. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

    PubMed

    Snowden, Christopher J; Thomas, Benjamin; Baxter, Charles J; Smith, J Andrew C; Sweetlove, Lee J

    2015-03-01

    Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

  5. Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

    PubMed

    Otto, H; Marti, T; Holz, M; Mogi, T; Stern, L J; Engel, F; Khorana, H G; Heyn, M P

    1990-02-01

    Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple

  6. Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base

    SciTech Connect

    Otto, H.; Marti, T.; Holz, M.; Mogi, T.; Stern, L.J.; Engel, F.; Khorana, H.G.; Heyn, M.P. )

    1990-02-01

    Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-((3-cholamidopropyl)dimethylammonio)-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base.

  7. Genome-wide association study and meta-analysis find that over 40 loci affect risk of type 1 diabetes.

    PubMed

    Barrett, Jeffrey C; Clayton, David G; Concannon, Patrick; Akolkar, Beena; Cooper, Jason D; Erlich, Henry A; Julier, Cécile; Morahan, Grant; Nerup, Jørn; Nierras, Concepcion; Plagnol, Vincent; Pociot, Flemming; Schuilenburg, Helen; Smyth, Deborah J; Stevens, Helen; Todd, John A; Walker, Neil M; Rich, Stephen S

    2009-06-01

    Type 1 diabetes (T1D) is a common autoimmune disorder that arises from the action of multiple genetic and environmental risk factors. We report the findings of a genome-wide association study of T1D, combined in a meta-analysis with two previously published studies. The total sample set included 7,514 cases and 9,045 reference samples. Forty-one distinct genomic locations provided evidence for association with T1D in the meta-analysis (P < 10(-6)). After excluding previously reported associations, we further tested 27 regions in an independent set of 4,267 cases, 4,463 controls and 2,319 affected sib-pair (ASP) families. Of these, 18 regions were replicated (P < 0.01; overall P < 5 × 10(-8)) and 4 additional regions provided nominal evidence of replication (P < 0.05). The many new candidate genes suggested by these results include IL10, IL19, IL20, GLIS3, CD69 and IL27. PMID:19430480

  8. Atmospheric Science Program (ASP) Data Archive

    DOE Data Explorer

    The Department of Energy's Atmospheric Science Program (ASP) originally consisted of an atmospheric chemistry program, an environmental meteorology program, a tropospheric aerosol program, and NARSTO activities. In 2004, the ASP was reconfigured to focus on aerosol radiative forcing of climate change: aerosol formation and evolution and aerosol properties that affect direct and indirect influences on climate and climate change. This included developing a comprehensive understanding of the atmospheric processes that control the transport, transformation, and fate of energy related trace chemicals and particulate matter. The current focus of the program is aerosol radiative forcing of climate. Effective October 1, 2009, The ASP merged with the Atmospheric Radiation Measurement Program (ARM), with the overall program now called Atmospheric System Research. The overall research goal is one that was shared in common, i.e. to further the understanding of how the climate, as a system works, and to represent the understanding in computer models. The Office of Science and Brookhaven announced, ôA major benefit of the merge is expected to be a strengthening of the aerosol- and cloud-related research components of the programs by bringing together the ARM capabilities of continuous remote sensing measurements of cloud properties and aerosol influences on radiation with the ASP capabilities for in-situ characterization of aerosol properties, evolution, and cloud interactions.ö [http://www.asp.bnl.gov/#New] The ASP data archive has now been moved to a new location in order to be maintained with ARM data. The new url is http://iop.archive.arm.gov/arm-iop/0special-data/ASP_Campaigns_past/. BNL continues to maintain an excellent list of ASP-publications at http://www.asp.bnl.gov/asp_pubs.html

  9. Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.

    PubMed

    Zhang, Jennifer; Katta, Viswanatham

    2012-01-01

    Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

  10. Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base

    SciTech Connect

    Needleman, R.; Chang, M.; Ni, B.; Varo, G.; Fornes, J.; White, S.H.; Lanyi, J.K. )

    1991-06-25

    The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. x-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.

  11. Parameter uncertainty for ASP models

    SciTech Connect

    Knudsen, J.K.; Smith, C.L.

    1995-10-01

    The steps involved to incorporate parameter uncertainty into the Nuclear Regulatory Commission (NRC) accident sequence precursor (ASP) models is covered in this paper. Three different uncertainty distributions (i.e., lognormal, beta, gamma) were evaluated to Determine the most appropriate distribution. From the evaluation, it was Determined that the lognormal distribution will be used for the ASP models uncertainty parameters. Selection of the uncertainty parameters for the basic events is also discussed. This paper covers the process of determining uncertainty parameters for the supercomponent basic events (i.e., basic events that are comprised of more than one component which can have more than one failure mode) that are utilized in the ASP models. Once this is completed, the ASP model is ready to be utilized to propagate parameter uncertainty for event assessments.

  12. The Future of the ASP Conference Series

    NASA Astrophysics Data System (ADS)

    Jensen, Joseph B.; Barnes, Jonathan; Moody, J. Ward; Szkody, Paula

    The Astronomical Society of the Pacific (ASP) has been publishing the proceedings of conferences in astronomy and astrophysics for more than 20 years. The ASP Conference Series (ASPCS) is widely known for its affordable and high quality printed volumes. The ASPCS is adapting to the changing market by making electronically published volumes available to subscribers around the world, including papers in the Astrophysics Data System (ADS) database, and allowing authors to post papers on e-print archives. We discuss the role of the printed book in our future plans, and how electronic publishing affects the types of products and services we offer. Recently there has been increasing pressure in the academic world for open access (electronic copies of scholarly publications made freely-available immediately after publication), and we discuss how the ASPCS is responding to the needs of the professional astronomical community, the ASP, and humanity at large. While we cannot yet provide full open access and stay in business, we are actively pursuing several initiatives to improve the quality of our product and the impact of the papers we publish.

  13. The Absolute Spectrum Polarimeter (ASP)

    NASA Technical Reports Server (NTRS)

    Kogut, A. J.

    2010-01-01

    The Absolute Spectrum Polarimeter (ASP) is an Explorer-class mission to map the absolute intensity and linear polarization of the cosmic microwave background and diffuse astrophysical foregrounds over the full sky from 30 GHz to 5 THz. The principal science goal is the detection and characterization of linear polarization from an inflationary epoch in the early universe, with tensor-to-scalar ratio r much greater than 1O(raised to the power of { -3}) and Compton distortion y < 10 (raised to the power of{-6}). We describe the ASP instrument and mission architecture needed to detect the signature of an inflationary epoch in the early universe using only 4 semiconductor bolometers.

  14. Human caspase-3 inhibition by Z-tLeu-Asp-H: tLeu(P{sub 2}) counterbalances Asp(P{sub 4}) and Glu(P{sub 3}) specific inhibitor truncation

    SciTech Connect

    Colantonio, Patrizia; Leboffe, Loris; Bolli, Alessandro; Marino, Maria; Ascenzi, Paolo; Luisi, Grazia

    2008-12-19

    Caspase-3 is responsible for the cleavage of several proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Designed on the cleavage site of PARP, Ac-Asp-Glu-Val-Asp-H has been reported as a highly specific inhibitor. To overcome the susceptibility to proteolysis, the intrinsic instability, and the scarce membrane permeability of tetra-peptidyl aldehydes, di- and tri-peptidyl caspase-3 inhibitors have been synthesized. Here, the synthesis and the inhibition properties of peptidyl aldehydes Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H are reported. Z-tLeu-Asp-H, Z-tLeu-Val-Asp-H, and Z-Val-tLeu-Asp-H inhibit competitively human caspase-3 activity in vitro with K{sub i}{sup 0} = 3.6 nM, 18.2 nM, and 109 nM, respectively (pH 7.4 and 25 deg. C). Moreover, Z-tLeu-Asp-H impairs apoptosis in human DLD-1 colon adenocarcinoma cells without affecting caspase-8. Therefore, Ac-Asp-Glu-Val-Asp-H can be truncated to Z-tLeu-Asp-H retaining nanomolar inhibitory activity in vitro and displaying action in whole cells, these properties reflect the unprecedented introduction of the bulky and lipophilic tLeu residue at the P{sub 2} position.

  15. Inflammatory markers and adipokines alter adipocyte-derived ASP production through direct and indirect immune interaction.

    PubMed

    Lu, H; Gauvreau, D; Tom, F-Q; Lapointe, M; Luo, X P; Cianflone, K

    2013-04-01

    Obesity and related metabolic diseases are associated with chronic low-grade inflammation, characterized by increased pro-inflammatory proteins. Several studies have demonstrated increases in acylation stimulating protein (ASP) and its precursor protein C3 in obesity, diabetes and dyslipidemia. To evaluate the effects of acute inflammatory factors and adipokines on ASP production and potential mechanisms of action, 3T3-L1 adipocytes were treated for 24 h with adipokines, cytokines, macrophage-conditioned media and direct co-culture with J774 macrophages. ASP and C3 in the media were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride stores). Leptin, adiponectin, IL-10, LPS and TNF-α increased ASP production (151%, 153%, 190%, 318%, 134%, P<0.05, respectively,). C5a and RANTES (Regulated and normal T cell expressed and secreted) decreased ASP production ( - 34%, - 47%, P<0.05), which was also associated with a decrease in the precursor protein C3 ( - 39% to - 51%, P<0.01), while keratinocyte chemoattractant (KC; murine IL-8 ortholog) had no effect on ASP and C3 secretion. By contrast, apelin, omentin and visfatin also decreased ASP ( - 27%, - 49%, - 22%, P<0.05), but without changes in precursor protein C3 secretion. Macrophage-conditioned media alone had little effect on C3 or ASP, while co-culture of adipocytes with macrophages markedly increased ASP and C3 production (272%, 167%, P<0.05). These in vitro results suggest various metabolic hormones and inflammatory factors can affect ASP production through increased precursor C3 production and/or by changing the rate of C3 conversion to ASP. As an adipokine, ASP could constitute a new link between adipocytes and macrophages.

  16. Review of Clinical Profile of IDegAsp.

    PubMed

    Unnikrishnan, A G; Singh, Awadhesh Kumar; Modi, K D; Saboo, Banshi; Garcha, Shilpa Chugh; Rao, Paturi Vishnupriya

    2015-05-01

    In patients with diabetes, treatment intensification requires basal and bolus insulin injections to control the fasting and prandial insulin needs. To overcome the burden of multiple daily injections, co-formulating basal and bolus insulins in single injection could allow a simple regimen with fewer injections. Current premixed insulin analogues are limited by the protaminated insulin component, which cannot provide effective basal coverage. While, long-acting insulin analogues like insulin glargine and insulin detemir cannot be combined with rapid-acting insulin analogues due to physicochemical incompatibility. Insulin degludec/insulin aspart (IDegAsp) is a soluble co-formulation of two distinct insulin analogues in the ratio of 70% ultra-long-acting insulin degludec (IDeg) and 30% rapid-acting insulin aspart (IAsp). The distinct PK/PD properties of IDeg and IAsp components are preserved in the co-formulation, with the rapid absorption characteristics of IAsp and flat and stable profile of IDeg maintained separately. Size exclusion chromatography studies of IDegAsp indicate that IDeg and lAsp exist as stable di-hexamers and hexamers, respectively in the formulation. Moreover, at steady state, the prandial and basal glucose lowering effects of IDeg and IAsp were distinct and clearly separated. A clear dose-response relationship was observed in patients with type 1 and type 2 diabetes treated with IDegAsp. The glucose lowering effects of basal and prandial components of IDegAsp are maintained in elderly (≥ 65 years of age) patients with type 1 diabetes. In addition, the PK and clearance of IDeg and IAsp are not affected by mild, moderate or severe renal or hepatic impairment. Presence of two distinct insulin analogues, as a soluble co-formulation with basal component with an ultra-long duration of action makes IDegAsp an advance to premix insulins. PMID:26548030

  17. Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Ca(v)3.2 T-type channel.

    PubMed

    Park, Hyun-Jee; Park, So-Jung; Ahn, Eun-Joo; Lee, So-Young; Seo, Haengsoo; Lee, Jung-Ha

    2013-09-01

    Voltage-activated Ca2+ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca2+ selective filters of all high-voltage-activated Ca2+ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca2+ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca(v)3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca(v)3.2, including relative permeability between Ba2+ and Ca2+, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd2+ blocking sensitivity, and pH effects, in distinctive ways.

  18. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  19. Effect of Asp 96 isomerization on the properties of a lens αB-crystallin-derived short peptide.

    PubMed

    Takata, Takumi; Fujii, Noriko

    2015-12-10

    One of the major reasons for age-related cataract formation is an accumulation of insoluble lens proteins. In particular, higher-order α-crystallin aggregates, comprising αA and αB subunits, are insolubilized by the build up of various post-translational modifications over time. Although we previously found an exceptional amount of Asp96 isomerization in αB-crystallin from aged human lens, the biological effect remains unknown. To approximate the effect of Asp 96 isomerization in αB-crystallin, here residues 93-103 of αB-crystallin were chemically synthesized as peptides in which l-α-Asp was replaced with l-β-Asp, D-α-Asp, or D-β-Asp. The resulting peptides were then compared in a biological assay. The results showed that isomerization of Asp 96 altered both the local structure of peptide and its stability against enzymatic digestion. In addition, the synthesized peptides decreased the insoluble fraction of heated α-crystallin. The D-β-Asp-containing peptide further decreased heat-induced precipitation of α-crystallin, and a chaperone assay based on heated alcohol dehydrogenase implied differential interaction of the peptides with substrate depending on the Asp isomer present in each. Our results suggest that the formation of Asp isomers is likely to affect the higher-order oligomer structure of α-crystallin and thereby its chaperone functions in aged lens.

  20. The Annular Suspension and Pointing System /ASPS/

    NASA Technical Reports Server (NTRS)

    Anderson, W. W.; Woolley, C. T.

    1978-01-01

    The Annular Suspension and Pointing System (ASPS) may be attached to a carrier vehicle for orientation, mechanical isolation, and fine pointing purposes applicable to space experiments. It has subassemblies for both coarse and vernier pointing. A fourteen-degree-of-freedom simulation of the ASPS mounted on a Space Shuttle has yielded initial performance data. The simulation describes: the magnetic actuators, payload sensors, coarse gimbal assemblies, control algorithms, rigid body dynamic models of the payload and Shuttle, and a control system firing model.

  1. Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein.

    PubMed

    Zhang, Jennifer; Yip, Holly; Katta, Viswanatham

    2011-03-15

    A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in -18Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp-Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H(2)(18)O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without (18)O incorporation. The light chain peptide L2 VTITCITSTDID(12)DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp-Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD(24)DFK. No other isomerizations were detected in CDR peptides containing either Asp-Ser or Asp-Thr motifs.

  2. Saphire models and software for ASP evaluations

    SciTech Connect

    Sattison, M.B.

    1997-02-01

    The Idaho National Engineering Laboratory (INEL) over the three years has created 75 plant-specific Accident Sequence Precursor (ASP) models using the SAPHIRE suite of PRA codes. Along with the new models, the INEL has also developed a new module for SAPHIRE which is tailored specifically to the unique needs of ASP evaluations. These models and software will be the next generation of risk tools for the evaluation of accident precursors by both the U.S. Nuclear Regulatory Commission`s (NRC`s) Office of Nuclear Reactor Regulation (NRR) and the Office for Analysis and Evaluation of Operational Data (AEOD). This paper presents an overview of the models and software. Key characteristics include: (1) classification of the plant models according to plant response with a unique set of event trees for each plant class, (2) plant-specific fault trees using supercomponents, (3) generation and retention of all system and sequence cutsets, (4) full flexibility in modifying logic, regenerating cutsets, and requantifying results, and (5) user interface for streamlined evaluation of ASP events. Future plans for the ASP models is also presented.

  3. The ASP: Programs to Inspire Educators

    NASA Astrophysics Data System (ADS)

    Hurst, Anna; Gurton, S.; Bennett, M.; Berendson, M.; Gibbs, M.

    2006-12-01

    The Astronomical Society of the Pacific (ASP) provides educators with new approaches to hands-on astronomy and space science. Through interactive educational programs, our goal is to help more people understand, appreciate, and enjoy astronomy and science. Over the past several years, the ASP has re-dedicated itself to achieving this mission through an ever-expanding portfolio of programs. Our astronomy and education programs target educators of all descriptions classroom teachers, informal science educators (in science museums, planetariums, nature centers, etc.), college astronomy teachers, and amateur astronomers providing them with materials and training to capture the attention of their students and audiences and to introduce them to science via an initial engagement in astronomy. In this poster we provide an overview of current programs that include partnerships with the National Optical Astronomy Observatory, the Association of Science-Technology Centers, TERC, the Astronomical League, NASA, and the SETI Institute to address this broad range of formal and informal educators. Additionally, the poster will provide a summary of recently conducted research by the ASP regarding the Project ASTRO program, done in cooperation with our national partners, to gauge whether the program, as perceived by the teachers participating in Project ASTRO, a) assists in correcting common misconceptions in astronomy or science and b) improve students' attitudes towards science. Additional information regarding the ASP's educational programs can be found at: www.astrosociety.org/education.html

  4. Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target

    PubMed Central

    Cox, Jonathan A. G.; Fütterer, Klaus; Abrahams, Katherine A.; Bhatt, Apoorva; Alderwick, Luke J.; Reynolds, Robert C.; Loman, Nicholas J.; Nataraj, VijayaShankar; Alemparte, Carlos; Barros, David; Lloyd, Adrian J.; Ballell, Lluis; Hobrath, Judith V.; Besra, Gurdyal S.

    2014-01-01

    The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å. PMID:25409504

  5. Acute injection of ASP in the third ventricle inhibits food intake and locomotor activity in rats.

    PubMed

    Roy, Christian; Roy, Marie-Claude; Gauvreau, Danny; Poulin, Anne-Marie; Tom, Fun-Qun; Timofeeva, Elena; Richard, Denis; Cianflone, Katherine

    2011-07-01

    Acylation-stimulating protein (ASP; also known as C3adesArg) stimulates triglyceride synthesis and glucose transport via interaction with its receptor C5L2, which is expressed peripherally (adipose tissue, muscle) and centrally. Previous studies have shown that ASP-deficient mice (C3KO) and C5L2-deficient mice (C5L2KO) are hyperphagic (59 to 229% increase, P < 0.0001), which is counterbalanced by increased energy expenditure measured as oxygen consumption (Vo(2)) and a lower RQ. The aim of the present study was to evaluate ASP's effect on food intake, energy expenditure, and neuropeptide expression. Male rats were surgically implanted with intracerebroventricular (icv) cannulas directed toward the third ventricle. After a 5-h fast, rats were injected, and food intake was assessed at 0.5, 1, 2, 4, 16, 24, and 48 h, with a 5- to 7-day washout period between each injection. Acute icv injections of ASP (0.3-1,065 pmol) had a time-dependent effect on decreasing food intake by 20 to 57% (P < 0.05). Decreases were detected by 30 min (maximum 57%, P < 0.01) and at the highest dose effects extended to 48 h (19%, P < 0.05, 24- to 48-h period). Daily body weight gain was decreased by 131% over the first 24 h and 29% over the second 24 h (P < 0.05). A conditioned taste aversion test indicated that there was no malaise. Furthermore, acute ASP injection affected energy substrate usage, demonstrated by decreased Vo(2) and RQ (P < 0.05; implicating greater fatty acid usage), with a 49% decrease in total activity over 24 h (P < 0.05). ASP administration also increased anorexic neuropeptide POMC expression (44%) in the arcuate nucleus, with no change in NPY. Altogether ASP may have central in addition to peripheral effects.

  6. SAPHIRE models and software for ASP evaluations

    SciTech Connect

    Sattison, M.B.; Schroeder, J.A.; Russell, K.D.

    1995-04-01

    The Idaho National Engineering Laboratory (INEL) over the past year has created 75 plant-specific Accident Sequence Precursor (ASP) models using the SAPHIRE suite of PRA codes. Along with the new models, the INEL has also developed a new module for SAPHIRE which is tailored specifically to the unique needs of conditional core damage probability (CCDP) evaluations. These models and software will be the next generation of risk tools for the evaluation of accident precursors by both NRR and AEOD. This paper presents an overview of the models and software. Key characteristics include: (1) classification of the plant models according to plant response with a unique set of event trees for each plant class, (2) plant-specific fault trees using supercomponents, (3) generation and retention of all system and sequence cutsets, (4) full flexibility in modifying logic, regenerating cutsets, and requantifying results, and (5) user interface for streamlined evaluation of ASP events.

  7. Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp.

    PubMed

    Calabrese, Antonio N; Markulic, Katarina; Musgrave, Ian F; Guo, Hui; Zhang, Lixin; Bowie, John H

    2012-12-01

    The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH(2))] and citropin 1.1 [GLFDVIKKVASVIGGL(NH(2))] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d(3)/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10(-9)M) but no different from the Asp isomer at concentrations>10(-9) M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.

  8. New technique for fertilizing eggs of burbot, asp and ide under hatchery conditions.

    PubMed

    Kucharczyk, Dariusz; Nowosad, Joanna; Łuczyński, Marek J; Targońska, Katarzyna

    2016-09-01

    The development of a new protocol for egg fertilization may increase embryo survival and benefit the aquaculture process. In the present study, a new technique of partially adding sperm to activated eggs in the artificial fertilization of burbot (Lota lota), ide (Leuciscus idus) and asp (Aspius aspius) eggs was evaluated. If the same volume of sperm was divided into two or three parts and added to eggs in 30-60s intervals, it significantly improved embryo survival at the eyed-egg-stage of development. In the present study, the periodic addition of spermatozoa to eggs affected fertilization (ide and asp) and embryo survival rates (ide, asp and burbot) and might be successfully applied under hatchery conditions. PMID:27470201

  9. Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

    SciTech Connect

    Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2013-02-15

    Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.

  10. Electronic trigger for the ASP experiment

    SciTech Connect

    Wilson, R.J.

    1985-11-01

    The Anomalous Single Photon (ASP) electronic trigger is described. The experiments is based on an electromagnetic calorimeter composed of arrays of lead glass blocks, read out with photo-multiplier tubes, surrounding the interaction point at the PEP storage ring. The primary requirement of the trigger system is to be sensitive to low energy (approx. =0.5 GeV and above) photons whilst discriminating against high backgrounds at PEP. Analogue summing of the PMT signals and a sequence of programmable digital look-up tables produces a ''dead-timeless'' trigger for the beam collision rate of 408 kHz. 6 refs., 6 figs.

  11. Expression profiling of muscle reveals transcripts differentially expressed in muscle that affect water-holding capacity of pork.

    PubMed

    Ponsuksili, Siriluck; Murani, Eduard; Phatsara, Chirawath; Jonas, Elisabeth; Walz, Christina; Schwerin, Manfred; Schellander, Karl; Wimmers, Klaus

    2008-11-12

    To identify biological processes as well as molecular markers for drip loss, a parameter for water holding capacity of meat, the M. longissimus dorsi transcriptomes of six divergent sib pairs were analyzed using Affymetrix Porcine Genome Array. Functional categories of differentially regulated transcripts were determined by single-gene analysis and gene set analysis. The transcripts being up-regulated at high drip loss belong to groups of genes functionally categorized as genes of membrane proteins, signal transduction, cell communication, response to stimulus, and cytoskeleton. Among genes down-regulated with high drip loss, functional groups of oxidoreductase activity, lipid metabolism, and electron transport were identified. Differential regulation of the abundance of transcripts of these biological networks in live muscle affect mortem biochemical processes of meat maturation. Knowledge of this functional link is indicative for the identification of candidate genes for improvement of meat quality. PMID:18922009

  12. Isomerization and epimerization of the aspartyl tetrapeptide Ala-Phe-Asp-GlyOH at pH 10-A CE study.

    PubMed

    Brückner, Christin; Bunz, Svenja-Catharina; Imhof, Diana; Neusüss, Christian; Scriba, Gerhard K E

    2013-09-01

    Isomerization and enantiomerization of Asp in the tetrapeptide Ala-Phe-Asp-GlyOH are studied at pH 10 and 80°C as well as 25°C. CE-MS allowed the distinction between α-Asp and β-Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfey's reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order Asp > Ala > Phe. The CE assay was validated with respect to linearity, LOQ, LOD, and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β-Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half-life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.

  13. 30 CFR 250.1921 - What qualifications must the ASP meet?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 2 2014-07-01 2014-07-01 false What qualifications must the ASP meet? 250.1921... Environmental Management Systems (SEMS) § 250.1921 What qualifications must the ASP meet? (a) The ASP must meet....198) or its equivalent; (b) The ASP must be accredited by a BSEE-approved AB; and (c) The ASP...

  14. 30 CFR 250.1921 - What qualifications must the ASP meet?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 2 2013-07-01 2013-07-01 false What qualifications must the ASP meet? 250.1921... Environmental Management Systems (SEMS) § 250.1921 What qualifications must the ASP meet? (a) The ASP must meet....198) or its equivalent; (b) The ASP must be accredited by a BSEE-approved AB; and (c) The ASP...

  15. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    PubMed

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

  16. Highly Significant Linkage to the SLI1 Locus in an Expanded Sample of Individuals Affected by Specific Language Impairment

    PubMed Central

    2004-01-01

    Specific language impairment (SLI) is defined as an unexplained failure to acquire normal language skills despite adequate intelligence and opportunity. We have reported elsewhere a full-genome scan in 98 nuclear families affected by this disorder, with the use of three quantitative traits of language ability (the expressive and receptive tests of the Clinical Evaluation of Language Fundamentals and a test of nonsense word repetition). This screen implicated two quantitative trait loci, one on chromosome 16q (SLI1) and a second on chromosome 19q (SLI2). However, a second independent genome screen performed by another group, with the use of parametric linkage analyses in extended pedigrees, found little evidence for the involvement of either of these regions in SLI. To investigate these loci further, we have collected a second sample, consisting of 86 families (367 individuals, 174 independent sib pairs), all with probands whose language skills are ⩾1.5 SD below the mean for their age. Haseman-Elston linkage analysis resulted in a maximum LOD score (MLS) of 2.84 on chromosome 16 and an MLS of 2.31 on chromosome 19, both of which represent significant linkage at the 2% level. Amalgamation of the wave 2 sample with the cohort used for the genome screen generated a total of 184 families (840 individuals, 393 independent sib pairs). Analysis of linkage within this pooled group strengthened the evidence for linkage at SLI1 and yielded a highly significant LOD score (MLS = 7.46, interval empirical P<.0004). Furthermore, linkage at the same locus was also demonstrated to three reading-related measures (basic reading [MLS = 1.49], spelling [MLS = 2.67], and reading comprehension [MLS = 1.99] subtests of the Wechsler Objectives Reading Dimensions). PMID:15133743

  17. Simultaneous stereoinversion and isomerization at the Asp-4 residue in βB2-crystallin from the aged human eye lenses.

    PubMed

    Fujii, Norihiko; Kawaguchi, Takehiro; Sasaki, Hiroshi; Fujii, Noriko

    2011-10-11

    The lens proteins are composed of α-, β-, and γ-crystallins that interact with each other to maintain the transparency and refractive power of the lens. Because the lens crystallins are long-lived proteins, they undergo various post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation. In βB2-crystallin, which is the most abundant β-crystallin, the deamidation of asparagine and glutamine residues has been reported. Here, we found that the aspartyl (Asp) residue at position 4 of βB2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon residue D-β-Asp. Surprisingly, the D/L ratio of β-Asp at position 4 in βB2-crystallin from elderly donors (67-77 year old) was 0.88-3.21. A D/L ratio of amino acids greater than 1.0 is defined as an inversion of configuration from the L- to D-form, rather than a racemization. These extremely high D/L ratios are equivalent to those of Asp-58 and Asp-151 (D/L ratio: 3.1 for Asp-58 and 5.7 for Asp-151) in αA-crystallin from elderly donors (~80 year old) as reported previously. Initially, we identified specific Asp residues in the β-crystallin family of proteins that undergo a high degree of inversion. These results show that the isomerization and inversion of Asp residues occurs both in the α- and β-crystallins of the lens. Inversion of these Asp residues directly affects the higher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions and disrupt the function of crystallins in the lens.

  18. Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not.

    PubMed

    Singh, Pawan; Tripathi, Pankaj; Muniyappa, K

    2010-01-01

    Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active

  19. Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin

    SciTech Connect

    Yamazaki, Yoichi; Hatanka, Minoru; Kandori, Hideki

    1995-05-30

    Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm{sup -1}, change into stronger H-bonding states in L of the wild type. Thr46{r_arrow}Val substitution abolished these bands in spite of the fact that [3-{sup 18}O]threonine-labeling did not shift with them indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96{r_arrow}Asn substitution in Thr46{r_arrow}Val/Asp96{r_arrow}Asn. A single Asp96{r_arrow}Asn substitution abolished the 3607 cm{sup -1} band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two of vibrational bands of the Schiff base mode at 1312 and 1301 cm{sup -1} and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecule sin the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and the affect interaction of the Schiff base with Asp 85, that is far removed from this region. 32 refs., 7 figs.

  20. Methods improvements incorporated into the SAPHIRE ASP models

    SciTech Connect

    Sattison, M.B.; Blackman, H.S.; Novack, S.D.

    1995-04-01

    The Office for Analysis and Evaluation of Operational Data (AEOD) has sought the assistance of the Idaho National Engineering Laboratory (INEL) to make some significant enhancements to the SAPHIRE-based Accident Sequence Precursor (ASP) models recently developed by the INEL. The challenge of this project is to provide the features of a full-scale PRA within the framework of the simplified ASP models. Some of these features include: (1) uncertainty analysis addressing the standard PRA uncertainties and the uncertainties unique to the ASP models and methods, (2) incorporation and proper quantification of individual human actions and the interaction among human actions, (3) enhanced treatment of common cause failures, and (4) extension of the ASP models to more closely mimic full-scale PRAs (inclusion of more initiators, explicitly modeling support system failures, etc.). This paper provides an overview of the methods being used to make the above improvements.

  1. Hereditary systemic amyloidosis due to Asp76Asn variant β2-microglobulin.

    PubMed

    Valleix, Sophie; Gillmore, Julian D; Bridoux, Frank; Mangione, Palma P; Dogan, Ahmet; Nedelec, Brigitte; Boimard, Mathieu; Touchard, Guy; Goujon, Jean-Michel; Lacombe, Corinne; Lozeron, Pierre; Adams, David; Lacroix, Catherine; Maisonobe, Thierry; Planté-Bordeneuve, Violaine; Vrana, Julie A; Theis, Jason D; Giorgetti, Sofia; Porcari, Riccardo; Ricagno, Stefano; Bolognesi, Martino; Stoppini, Monica; Delpech, Marc; Pepys, Mark B; Hawkins, Philip N; Bellotti, Vittorio

    2012-06-14

    We describe a kindred with slowly progressive gastrointestinal symptoms and autonomic neuropathy caused by autosomal dominant, hereditary systemic amyloidosis. The amyloid consists of Asp76Asn variant β(2)-microglobulin. Unlike patients with dialysis-related amyloidosis caused by sustained high plasma concentrations of wild-type β(2)-microglobulin, the affected members of this kindred had normal renal function and normal circulating β(2)-microglobulin values. The Asp76Asn β(2)-microglobulin variant was thermodynamically unstable and remarkably fibrillogenic in vitro under physiological conditions. Previous studies of β(2)-microglobulin aggregation have not shown such amyloidogenicity for single-residue substitutions. Comprehensive biophysical characterization of the β(2)-microglobulin variant, including its 1.40-Å, three-dimensional structure, should allow further elucidation of fibrillogenesis and protein misfolding. PMID:22693999

  2. Open Access and the Future of the ASP Conference Series

    NASA Astrophysics Data System (ADS)

    Jensen, J. B.; Moody, J. W.; Barnes, J.

    2010-10-01

    The Astronomical Society of the Pacific (ASP) has been publishing the proceedings of conferences in astronomy and astrophysics for more than twenty years. The ASP Conference Series (ASPCS) is widely known for its affordable and high-quality printed volumes. The ASPCS is adapting to the changing ways astronomers use our proceedings volumes, both electronically and in print. Recently there has been increasing pressure from government agencies and the academic community for "open access" (electronic copies of scholarly publications made freely available immediately after publication), and we discuss how the ASPCS is responding to the needs of the professional astronomical community, the scholarly society that supports us (the ASP), and humanity at large. While we cannot yet provide full open access and stay in business, we are actively pursuing several initiatives to improve the quality of our product and the impact of the papers we publish.

  3. Asp-52 in combination with Asp-398 plays a critical role in ATP hydrolysis of chaperonin GroEL.

    PubMed

    Koike-Takeshita, Ayumi; Mitsuoka, Kaoru; Taguchi, Hideki

    2014-10-24

    The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼ 150 h (∼ 6 days), providing a good model to characterize the football-shaped complex.

  4. Annular Suspension and Pointing System (ASPS) magnetic rotary joint

    NASA Technical Reports Server (NTRS)

    Smith, W. E.; Quach, W.; Thomas, W.

    1993-01-01

    The Annular Suspension and Pointing System (ASPS) is a prototype of flight hardware for a high-accuracy space payload pointing mount. The long term project objective is to perform modifications and implement improvements to the existing ASPS in hopes of recommission. Also, new applications will be investigated for this technology. This report will focus on the first aspect of this overall goal, to establish operation of a single bearing station. Presented is an overview of the system history and bearing operation followed by the processes, results, and status of the single bearing study.

  5. A rapid, comprehensive liquid chromatography-mass spectrometry (LC-MS)-based survey of the Asp isomers in crystallins from human cataract lenses.

    PubMed

    Fujii, Norihiko; Sakaue, Hiroaki; Sasaki, Hiroshi; Fujii, Noriko

    2012-11-16

    Cataracts are caused by clouding of the eye lens and may lead to partial or total loss of vision. The mechanism of cataract development, however, is not well understood. It is thought that abnormal aggregates of lens proteins form with age, causing loss of lens clarity and development of the cataract. Lens proteins are composed of soluble α-, β-, and γ-crystallins, and as long lived proteins, they undergo post-translational modifications including isomerization, deamidation, and oxidation, which induce insolubilization, aggregation, and loss of function that may lead to cataracts. Therefore, analysis of post-translational modifications of individual amino acid residues in proteins is important. However, detection of the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only achieved using complex methodology. In this study, we describe a new method for the analysis of isomerization of individual Asp residues in proteins using LC-MS and the corresponding synthetic peptides containing the Asp isomers. This makes it possible to analyze isomers of Asp residues in proteins precisely and quickly. We demonstrate that Asp-58, -76, -84, and -151 of αA-crystallin and Asp-62 and -96 of αB-crystallin are highly converted to lβ-, dβ-, and dα-isomers. The amount of isomerization of Asp is greater in the insoluble fraction at all Asp sites in lens proteins, therefore indicating that isomerization of these Asp residues affects the higher order structure of the proteins and contributes to the increase in aggregation, insolubilization, and disruption of function of proteins in the lens, leading to the cataract.

  6. Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences.

    PubMed

    Shanmugam, Raghuvaran; Fierer, Jacob; Kaiser, Steffen; Helm, Mark; Jurkowski, Tomasz P; Jeltsch, Albert

    2015-01-01

    The Dnmt2 RNA methyltransferase catalyses the methylation of C38 in the anticodon loop of tRNA-Asp, but the molecular role of this methylation is unknown. Here, we report that mouse aspartyl-tRNA synthetase shows a four to fivefold preference for C38-methylated tRNA-Asp. Consistently, a 30% reduced charging level of tRNA-Asp was observed in Dnmt2 knockout (KO) murine embryonic fibroblast cells. Gene expression analysis with fluorescent reporter proteins fused to an N-terminal poly-Asp sequence showed that protein synthesis of poly-Asp-tagged reporter proteins was reduced in Dnmt2 KO cells as well. The same effect was observed with endogenous proteins containing poly-Asp sequences, indicating that Dnmt2-mediated C38 methylation of tRNA-Asp regulates the translation of proteins containing poly-Asp sequences. Gene ontology searches for proteins containing poly-Asp sequences in the human proteome showed that a significant number of these proteins have roles in transcriptional regulation and gene expression. Hence, the Dnmt2-mediated methylation of tRNA-Asp exhibits a post-transcriptional regulatory role by controlling the synthesis of a group of target proteins containing poly-Asp sequences.

  7. Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences

    PubMed Central

    Shanmugam, Raghuvaran; Fierer, Jacob; Kaiser, Steffen; Helm, Mark; Jurkowski, Tomasz P; Jeltsch, Albert

    2015-01-01

    The Dnmt2 RNA methyltransferase catalyses the methylation of C38 in the anticodon loop of tRNA-Asp, but the molecular role of this methylation is unknown. Here, we report that mouse aspartyl-tRNA synthetase shows a four to fivefold preference for C38-methylated tRNA-Asp. Consistently, a 30% reduced charging level of tRNA-Asp was observed in Dnmt2 knockout (KO) murine embryonic fibroblast cells. Gene expression analysis with fluorescent reporter proteins fused to an N-terminal poly-Asp sequence showed that protein synthesis of poly-Asp-tagged reporter proteins was reduced in Dnmt2 KO cells as well. The same effect was observed with endogenous proteins containing poly-Asp sequences, indicating that Dnmt2-mediated C38 methylation of tRNA-Asp regulates the translation of proteins containing poly-Asp sequences. Gene ontology searches for proteins containing poly-Asp sequences in the human proteome showed that a significant number of these proteins have roles in transcriptional regulation and gene expression. Hence, the Dnmt2-mediated methylation of tRNA-Asp exhibits a post-transcriptional regulatory role by controlling the synthesis of a group of target proteins containing poly-Asp sequences. PMID:27462411

  8. Right Ventricular Adaptation Is Associated with the Glu298Asp Variant of the NOS3 Gene in Elite Athletes.

    PubMed

    Szelid, Zsolt; Lux, Árpád; Kolossváry, Márton; Tóth, Attila; Vágó, Hajnalka; Lendvai, Zsuzsanna; Kiss, Loretta; Maurovich-Horvat, Pál; Bagyura, Zsolt; Merkely, Béla

    2015-01-01

    Nitric oxide (NO), an important endogenous pulmonary vasodilator is synthetized by the endothelial NO synthase (NOS3). Reduced NO bioavailability and thus the Glu298Asp polymorphism of NOS3 may enhance right ventricular (RV) afterload and hypertrophic remodeling and influence athletic performance. To test this hypothesis world class level athletes (water polo players, kayakers, canoeists, rowers, swimmers, n = 126) with a VO2 maximum greater than 50ml/kg/min were compared with non-athletic volunteers (n = 155). Cardiopulmonary exercise tests and cardiac magnetic resonance imaging (cMRI) were performed to determine structural or functional changes. Genotype distribution of the NOS3 Glu298Asp polymorphism was not affected by gender or physical performance. Cardiac MRI showed increased stroke volume with eccentric hypertrophy in all athletes regardless of their genotype. However, the Asp allelic variant carriers had increased RV mass index (32±6g versus 27±6g, p<0.01) and larger RV stroke volume index (71±10ml versus 64±10ml, p<0.01) than athletes with a Glu/Glu genotype. Genotype was not significantly associated with athletic performance. In the non-athletic group no genotype related differences were detected. The association between the NOS3 Glu298Asp polymorphism and RV structure and dimension in elite athletes emphasizes the importance of NOS3 gene function and NO bioavailability in sport related cardiac adaptation. PMID:26517550

  9. Right Ventricular Adaptation Is Associated with the Glu298Asp Variant of the NOS3 Gene in Elite Athletes

    PubMed Central

    Kolossváry, Márton; Tóth, Attila; Vágó, Hajnalka; Lendvai, Zsuzsanna; Kiss, Loretta; Maurovich-Horvat, Pál; Bagyura, Zsolt; Merkely, Béla

    2015-01-01

    Nitric oxide (NO), an important endogenous pulmonary vasodilator is synthetized by the endothelial NO synthase (NOS3). Reduced NO bioavailability and thus the Glu298Asp polymorphism of NOS3 may enhance right ventricular (RV) afterload and hypertrophic remodeling and influence athletic performance. To test this hypothesis world class level athletes (water polo players, kayakers, canoeists, rowers, swimmers, n = 126) with a VO2 maximum greater than 50ml/kg/min were compared with non-athletic volunteers (n = 155). Cardiopulmonary exercise tests and cardiac magnetic resonance imaging (cMRI) were performed to determine structural or functional changes. Genotype distribution of the NOS3 Glu298Asp polymorphism was not affected by gender or physical performance. Cardiac MRI showed increased stroke volume with eccentric hypertrophy in all athletes regardless of their genotype. However, the Asp allelic variant carriers had increased RV mass index (32±6g versus 27±6g, p<0.01) and larger RV stroke volume index (71±10ml versus 64±10ml, p<0.01) than athletes with a Glu/Glu genotype. Genotype was not significantly associated with athletic performance. In the non-athletic group no genotype related differences were detected. The association between the NOS3 Glu298Asp polymorphism and RV structure and dimension in elite athletes emphasizes the importance of NOS3 gene function and NO bioavailability in sport related cardiac adaptation. PMID:26517550

  10. Fusion of Na-ASP-2 with human immunoglobulin Fcγ abrogates histamine release from basophils sensitized with anti-Na-ASP-2 IgE.

    PubMed

    Zhan, Bin; Santiago, H; Keegan, B; Gillespie, P; Xue, J; Bethony, J; de Oliveira, L M; Jiang, D; Diemert, D; Xiao, S-H; Jones, K; Feng, X; Hotez, P J; Bottazzi, M E

    2012-01-01

    Na-ASP-2 is a major protein secreted by infective third-stage larvae (L3) of the human hookworm Necator americanus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombinant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N. americanus. To prevent IgE-mediated allergic reactions induced by Na-ASP-2 but keep its immunogenicity as a vaccine antigen, we designed and tested a genetically engineered fusion protein, Fcγ/Na-ASP-2, composed of full-length Na-ASP-2 and truncated human IgG Fcγ1 that targets the negative signalling receptor FcγRIIb expressed on pro-allergic cells. The chimeric recombinant Fcγ/Na-ASP-2 protein was expressed in Pichia pastoris and shared the similar antigenicity as native Na-ASP-2. Compared to Na-ASP-2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti-Na-ASP-2 IgE obtained from individuals living in a hookworm-endemic area. In dogs infected with canine hookworm, Fcγ/Na-ASP-2 resulted in significantly reduced immediate-type skin reactivity when injected intradermally compared with Na-ASP-2. Hamsters vaccinated with Fcγ/Na-ASP-2 formulated with Alhydrogel(®) produced specific IgG that recognized Na-ASP-2 and elicited similar protection level against N. americanus L3 challenge as native Na-ASP-2.

  11. Influence of Lβ-, Dα- and Dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70-88 peptide.

    PubMed

    Fujii, Noriko; Fujii, Norihiko; Kida, Masashi; Kinouchi, Tadatoshi

    2010-11-01

    Proteins have been considered to consist exclusively of L-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the D-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of D- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to D-isomers in a protein, we synthesized peptides corresponding to the 70-88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which Lα-Asp was replaced with Lβ-Asp, Dα-Asp, and Dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (Lβ-Asp, Dα-Asp, and Dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing Lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.

  12. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Cation Mediation on Asp Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2012-01-01

    The binding of a negatively charged residue, aspartic acid (Asp) in tripeptide arginine-glycine-aspartic acid, onto a negatively charged hydroxylated rutile (110) surface in aqueous solution, containing divalent (Mg{sup 2+}, Ca{sup 2+}, or Sr{sup 2+}) or monovalent (Na{sup +}, K{sup +}, or Rb{sup +}) cations, was studied by molecular dynamics (MD) simulations. The results indicate that ionic radii and charges will significantly affect the hydration, adsorption geometry, and distance of cations from the rutile surface, thereby regulating the Asp/rutile binding mode. The adsorption strength of monovalent cations on the rutile surface in the order Na{sup +} > K{sup +} > Rb{sup +} shows a 'reverse' lyotropic trend, while the divalent cations on the same surface exhibit a 'regular' lyotropic behavior with decreasing crystallographic radii (the adsorption strength of divalent cations: Sr{sup 2+} > Ca{sup 2+} > Mg{sup 2+}). The Asp side chain in NaCl, KCl, and RbCl solutions remains stably H-bonded to the surface hydroxyls and the inner-sphere adsorbed compensating monovalent cations act as a bridge between the COO{sup -} group and the rutile, helping to 'trap' the negatively charged Asp side chain on the negatively charged surface. In contrast, the mediating divalent cations actively participate in linking the COO{sup -} group to the rutile surface; thus the Asp side chain can remain stably on the rutile (110) surface, even if it is not involved in any hydrogen bonds with the surface hydroxyls. Inner- and outer-sphere geometries are all possible mediation modes for divalent cations in bridging the peptide to the rutile surface.

  13. Structural elucidation of the DFG-Asp in and DFG-Asp out states of TAM kinases and insight into the selectivity of their inhibitors.

    PubMed

    Messoussi, Abdellah; Peyronnet, Lucile; Feneyrolles, Clémence; Chevé, Gwénaël; Bougrin, Khalid; Yasri, Aziz

    2014-10-10

    Structural elucidation of the active (DFG-Asp in) and inactive (DFG-Asp out) states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

  14. [Effect of point substitutions of Asp-714 and Asp-720 residues on the structure and function of the H+ -ATPase of the yeast plasma membrane].

    PubMed

    Petrov, V V; Ibragimov, R I

    2014-01-01

    Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H(+)-, Ca2(+)-, K(+)-, Na(+)-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H(+)-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino, acidreplacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The Asp714Asn point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression levels, activity, H+ transport, and ATP hydrolyzing activity. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.

  15. Effect of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on the morphology of the thymus in hypophysectomized young and old birds.

    PubMed

    Pateyk, A V; Baranchugova, L M; Rusaeva, N S; Obydenko, V I; Kuznik, B I

    2013-03-01

    Investigations were carried out on chicks of different age. It was found that the most pronounced changes in the morphology of the thymus occurred after neonatal hypophysectomy. These changes are least pronounced in old chicks. Peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthesized on the basis of amino acid composition of peptide complexes of the anterior and posterior pituitary lobes administered to hypophysectomized birds regardless of age promoted recovery of the morphological structures of the thymus. The anterior pituitary peptide (Lys-Glu-Asp-Gly) had more pronounced effect on the recovery of thymic structure than posterior pituitary peptide (Ala-Glu-Asp-Gly).

  16. ASP Performance Assessment: Toward a Science-Based Understanding

    SciTech Connect

    Sale, K

    2008-04-22

    Several approaches to ASP performance can be contemplated. Perhaps the ideal would be a full cost/benefit analysis (which is probably utterly infeasible). Another approach would be a test-based figure-of-merit (FOM), this approach has the virtue of being quantitative and the challenge that each customer and application would be characterized by a different FOM. The alternative proposed here is an approach that uses information about the limits of detection of real instruments to support informed judgments.

  17. Determination of ASPS performance for large payloads in the shuttle orbiter disturbance environment. [digital simulation

    NASA Technical Reports Server (NTRS)

    Keckler, C. R.; Kibler, K. S.; Powell, L. F.

    1979-01-01

    A high fidelity simulation of the annular suspension and pointing system (ASPS), its payload, and the shuttle orbiter was used to define the worst case orientations of the ASPS and its payload for the various vehicle disturbances, and to determine the performance capability of the ASPS under these conditions. The most demanding and largest proposed payload, the Solar Optical Telescope was selected for study. It was found that, in all cases, the ASPS more than satisfied the payload's requirements. It is concluded that, to satisfy facility class payload requirements, the ASPS or a shuttle orbiter free-drift mode (control system off) should be utilized.

  18. The modeling and design of the Annular Suspension and Pointing System /ASPS/. [for Space Shuttle

    NASA Technical Reports Server (NTRS)

    Kuo, B. C.; Lin, W. C. W.

    1979-01-01

    The Annular Suspension and Pointing System (ASPS) is a payload auxiliary pointing device of the Space Shuttle. The ASPS is comprised of two major subassemblies, a vernier and a coarse pointing subsystem. The three functions provided by the ASPS are related to the pointing of the payload, centering the payload in the magnetic actuator assembly, and tracking the payload mounting plate and shuttle motions by the coarse gimbals. The equations of motion of a simplified planar model of the ASPS are derived. Attention is given to a state diagram of the dynamics of the ASPS with position-plus-rate controller, the nonlinear spring characteristic for the wire-cable torque of the ASPS, the design of the analog ASPS through decoupling and pole placement, and the time response of different components of the continuous control system.

  19. Two-locus admixture linkage analysis of bipolar and unipolar affective disorder supports the presence of susceptibility loci on chromosomes 11p15 and 21q22

    SciTech Connect

    Smyth, C.; Kalsi, G.; O`Neill, J.

    1997-02-01

    Following a report of a linkage study that yielded evidence for a susceptibility locus for bipolar affective disorder on the long arm of chromosome 21, we studied 23 multiply affected pedigrees collected from Iceland and the UK, using the markers PFKL, D21S171, and D21S49. Counting only bipolar cases as affected, a two-point LOD of 1.28 was obtained using D21S171 ({theta} = 0.01, {alpha} = 0.35), with three Icelandic families producing LODs of 0.63, 0.62, and 1.74 (all at {theta} = 0.0). Affected sib pair analysis demonstrated increased allele sharing at D21S171 (P = 0.001) when unipolar cases were also considered affected. The same set of pedigrees had previously been typed for a tyrosine hydroxylase gene (TH) polymorphism at 11p15 and had shown some moderate evidence for linkage. When information from TH and the 21q markers was combined in a two-locus admixture analysis, an overall admixture LOD of 3.87 was obtained using the bipolar affection model. Thus the data are compatible with the hypothesis that a locus at or near TH influences susceptibility in some pedigrees, while a locus near D21S171 is active in others. Similar analyses in other datasets should be carried out to confirm or refute our tentative finding. 66 refs., 3 tabs.

  20. Septins AspA and AspC are important for normal development and limit the emergence of new growth foci in the multicellular fungus Aspergillus nidulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Septins are cytoskeletal proteins found in fungi, animals and microsporidia where they form multi-septin heteropolymeric complexes that act as scaffolds recruiting and organizing other proteins to ensure normal cell division and development. Here we characterize AspA and AspC, two of the five septin...

  1. The adjuvanticity of an O. volvulus-derived rOv-ASP-1 protein in mice using sequential vaccinations and in non-human primates.

    PubMed

    Wang, Jing; Tricoche, Nancy; Du, Lanying; Hunter, Meredith; Zhan, Bin; Goud, Gaddam; Didier, Elizabeth S; Liu, Jing; Lu, Lu; Marx, Preston A; Jiang, Shibo; Lustigman, Sara

    2012-01-01

    Adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. We previously reported that the Onchocerca volvulus recombinant protein rOv-ASP-1 can induce activation and maturation of naïve human DCs and therefore could be used as an innate adjuvant to promote balanced Th1 and Th2 responses to bystander vaccine antigens in mice. With a few vaccine antigens, it also promoted a Th1-biased response based on pronounced induction of Th1-associated IgG2a and IgG2b antibody responses and the upregulated production of Th1 cytokines, including IL-2, IFN-γ, TNF-α and IL-6. However, because it is a protein, the rOv-ASP-1 adjuvant may also induce anti-self-antibodies. Therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rOv-ASP-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. In this study, we have established rOv-ASP-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (RBD) of SARS-CoV spike protein and a commercial influenza virus hemagglutinin (HA) vaccine comprised of three virus strains. Moreover, the adjuvanticity of rOv-ASP-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rOv-ASP-1 antibodies was present in the sera of mice before the administration of the second vaccine. To further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (NHPs) with RBD plus rOv-ASP-1 and showed that rOv-ASP-1 could induce high titres of functional and protective anti-RBD antibody responses in NHPs. Notably, the rOv-ASP-1 adjuvant did not induce high titer antibodies against self in NHPs. Thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant.

  2. The adjuvanticity of an O. volvulus-derived rOv-ASP-1 protein in mice using sequential vaccinations and in non-human primates.

    PubMed

    Wang, Jing; Tricoche, Nancy; Du, Lanying; Hunter, Meredith; Zhan, Bin; Goud, Gaddam; Didier, Elizabeth S; Liu, Jing; Lu, Lu; Marx, Preston A; Jiang, Shibo; Lustigman, Sara

    2012-01-01

    Adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. We previously reported that the Onchocerca volvulus recombinant protein rOv-ASP-1 can induce activation and maturation of naïve human DCs and therefore could be used as an innate adjuvant to promote balanced Th1 and Th2 responses to bystander vaccine antigens in mice. With a few vaccine antigens, it also promoted a Th1-biased response based on pronounced induction of Th1-associated IgG2a and IgG2b antibody responses and the upregulated production of Th1 cytokines, including IL-2, IFN-γ, TNF-α and IL-6. However, because it is a protein, the rOv-ASP-1 adjuvant may also induce anti-self-antibodies. Therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rOv-ASP-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. In this study, we have established rOv-ASP-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (RBD) of SARS-CoV spike protein and a commercial influenza virus hemagglutinin (HA) vaccine comprised of three virus strains. Moreover, the adjuvanticity of rOv-ASP-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rOv-ASP-1 antibodies was present in the sera of mice before the administration of the second vaccine. To further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (NHPs) with RBD plus rOv-ASP-1 and showed that rOv-ASP-1 could induce high titres of functional and protective anti-RBD antibody responses in NHPs. Notably, the rOv-ASP-1 adjuvant did not induce high titer antibodies against self in NHPs. Thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant. PMID

  3. Paradoxical glucose-sensitizing yet proinflammatory effects of acute ASP administration in mice.

    PubMed

    Fisette, Alexandre; Poursharifi, Pegah; Oikonomopoulou, Katerina; Munkonda, Mercedes N; Lapointe, Marc; Cianflone, Katherine

    2013-01-01

    Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processes in vivo were evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-α in plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, the in vivo and in vitro experiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects.

  4. Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cui, Wei; Lapointe, Marc; Paglialunga, Sabina; Cianflone, Katherine

    2011-02-01

    In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

  5. eNOS Glu298Asp Polymorphism and Endothelial Dysfunction in Patients with and without End-stage Renal Disease

    PubMed Central

    İlhan, Nevin; Ateş, Kadir; İlhan, Necip; Kaman, Dilara; Çeliker, Hüseyin

    2016-01-01

    Background: Chronic kidney diseases are known to influence nitric oxide metabolites (NOx) and asymmetric dimethylarginine (ADMA), though the exact mechanism is still poorly understood. Aims: The purpose of the present study was to examine eNOS Glu298Asp gene polymorphism, plasma NOx and ADMA concentration in subjects with and without End-stage Renal Disease. Study Design: Case-control study. Methods: In this study, genotype distributions of Glu-298Asp in exon 7 of the eNOS gene polymorphisms in 130 hemodialysis and 64 peritoneal dialysis patients were compared with 92 controls. NOx was measured by using the Griess reaction while arginine, ADMA and SDMA measurements were performed by HPLC. Genotyping for eNOS Glu298Asp polymorphism was detected with the polymerase chain reaction and/or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: When the genotype frequencies of TT and GT genes were compared between both groups, there was no detected statistically important difference, even-though a TT genotype frequency was 27 (20.8%) versus 17 (26.6%), GT heterozygote genotype frequency was 52 (40%) versus 22 (34.4%), and GG homozygote genotype frequency was 51 (39.2%) versus 25 (39.1%), respectively (p>0.05). NOx, SDMA and ADMA concentrations were significantly elevated in subjects with hemodialysis patients as compared to their corresponding controls. Whereas nitrite was found to be significantly decreased in the patient with peritoneal dialysis. Conclusion: Not observed any connection between the Glu298Asp polymorphism in the eNOS gene and end-stage Renal Diseases in our study population under different dialysis treatments. However, higher ADMA and SDMA concentrations in subjects with ESRD support the existing hypothesis that NOx overproduction affects endothelial dysfunction. Thus, the reduction of ADMA and SDMA concentrations might play a protective role in ESRD patients. PMID:27403380

  6. Unexpected Enzyme TEM-126: Role of Mutation Asp179Glu

    PubMed Central

    Delmas, J.; Robin, F.; Bittar, F.; Chanal, C.; Bonnet, R.

    2005-01-01

    The clinical isolate Escherichia coli CF884 exhibited low-level resistance to ceftazidime (4 μg/ml) by a positive double-disk synergy test and apparent susceptibility to cefuroxime, cefotaxime, cefepime, cefpirome, and aztreonam. The enzyme implicated in this phenotype was a novel 180-kb plasmid-encoded TEM-type extended-spectrum β-lactamase designated TEM-126 which harbors the mutations Asp179Glu and Met182Thr. TEM-126 exhibited significant hydrolytic activity (kcat, 2 s−1) and a Km value of 82 μM against ceftazidime. Molecular dynamics simulations suggested that the substitution Asp179Glu induces subtle conformational changes to the omega loop which may favor the insertion of ceftazidime in the binding site and the correct positioning of the crucial residue Glu166. Overall, these results highlight the remarkable plasticity of TEM enzymes, which can expand their activity against ceftazidime by the addition of one carbon atom in the side chain of residue 179. PMID:16189109

  7. Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay.

    PubMed

    Bousché, O; Sonar, S; Krebs, M P; Khorana, H G; Rothschild, K J

    1992-12-01

    The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in

  8. C3 Polymorphism Influences Circulating Levels of C3, ASP and Lipids in Schizophrenic Patients.

    PubMed

    Nsaiba, Mohamed Jalloul; Lapointe, Marc; Mabrouk, Hajer; Douki, Wahiba; Gaha, Lotfi; Pérusse, Louis; Bouchard, Claude; Jrad, Besma Bel Hadj; Cianflone, Katherine

    2015-05-01

    Excessive activation of complement is associated with many diseases including schizophrenia. Investigation of C3 polymorphisms, circulating C3, cleavage product ASP/C3adesArg, and lipid metabolism. Cross-sectional analysis. C3 genotyping (CC vs GG for R102L) was performed on 434 Tunisian people consisting of 272 schizophrenic (SZ) patients and 162 control subjects. In a age- and gender-matched subgroups of the three genotypes (131 SZ and 112 NOR), plasma triglycerides, total cholesterol (C), LDL-C, HDL-C, ASP, and complement C3 were measured. C3 gene polymorphism influences BMI and plasma C3, ASP, triglyceride, total cholesterol, LDL-C and HDL-C among SZ patients (p < 0.05-0.0001), with increasing values demonstrated from CC (common form) to CG (heterozygote form) to GG (rare homozygote) forms. Significant correlations between plasma C3 and BMI, triglyceride, HDL-C and ASP (p < 0.05-0.0001) were observed, while ASP correlated with BMI and LDL-C (p = 0.005, p = 0.001, respectively) in SZ patients. Further, proportional conversion of C3 to ASP (%ASP/C3) also increased (p < 0.0001, GG>CG>CC). C3 polymorphisms and plasma C3, ASP and %ASP/C3 correlated with lipid parameters in this SZ population, suggesting that factors predisposing patients to schizophrenia are permissive for complement pathway activation and dyslipidemic influences.

  9. Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP.

    PubMed

    Jacobsen, Mette Dorph; Pedersen, Per Amstrup; Jorgensen, Peter Leth

    2002-02-01

    To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).

  10. 77 FR 16113 - ASP Ventures Corp., Order of Suspension of Trading

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-19

    ... From the Federal Register Online via the Government Publishing Office SECURITIES AND EXCHANGE COMMISSION ASP Ventures Corp., Order of Suspension of Trading March 15, 2012. It appears to the Securities... securities of ASP Ventures Corp. because it has not filed any periodic reports since the period...

  11. Characterization of virus strains resistant to the herpes virus helicase-primase inhibitor ASP2151 (Amenamevir).

    PubMed

    Chono, Koji; Katsumata, Kiyomitsu; Kontani, Toru; Shiraki, Kimiyasu; Suzuki, Hiroshi

    2012-08-15

    ASP2151 is an antiherpes agent targeting the helicase-primase complex of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV). We characterized the ASP2151-resistant HSV-1 and HSV-2 variants or mutants based on findings from sequencing analysis, growth, pathogenicity, and susceptibility testing, identifying several single base-pair substitutions resulting in amino acid changes in the helicase and primase subunit of ASP2151-resistant mutants. Amino acid alterations in the helicase subunit were clustered near helicase motif IV in the UL5 helicase gene of both HSV-1 and HSV-2, while the primase subunit substitution associated with reduced susceptibility, R367H, was found in ASP2151-resistant HSV-1 mutants. However, while susceptibility in the ASP2151-resistant HSV mutants to existing antiherpes agents was equivalent to that in wild-type HSV strains, ASP2151-resistant HSV mutants showed attenuated in vitro growth capability and in vivo pathogenicity compared with the parent strains. Taken together, our present findings demonstrated that important amino acid substitutions associated with reduced susceptibilities of HSV-1 and HSV-2 to ASP2151 exist in both the helicase and primase subunits of the helicase-primase complex, and that mutations in this complex against ASP2151 might confer defects in viral replication and pathogenicity.

  12. Absorption, distribution, metabolism and excretion of novel phosphodiesterase type 4 inhibitor ASP3258 in rats.

    PubMed

    Ohtsu, Yoshiaki; Sonoda, Takuya; Susaki, Yoko; Tohda, Toshifumi; Fukunaga, Yasuhisa; Iwatsubo, Takafumi; Noguchi, Kiyoshi

    2015-01-01

    The potent and selective phosphodiesterase 4 inhibitor ASP3258 is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease (COPD). After a single oral administration to rats, ASP3258 is rapidly absorbed with a bioavailability of 106%. In situ absorption data indicated that ASP3258 is mainly absorbed in the small intestine. Tissue distribution data after oral administration of (14)C-ASP3258 showed rapid and extensive distribution to various tissues. Excluding the gastrointestinal tract, the tissues with the highest concentrations were liver, heart and plasma. Liquid chromatography-nuclear magnetic resonance spectroscopy data revealed that O-glucuronidation of the carboxylic acid moiety of ASP3258 (formation of an acyl glucuronide) plays a key role in metabolism. No indication was found that the acyl glucuronide reacted with proteins in plasma or tissues. When (14)C-ASP3258 was orally administered to intact rats, urinary and fecal excretion accounted for 1.3% and 100.6% of the administered radioactivity, respectively. After a single oral administration of (14)C-ASP3258 to bile-cannulated rats, urinary and biliary excretion accounted for 0.7% and 93.8% of the administered radioactivity, respectively. These findings suggest that fecal excretion via bile plays an important role in the elimination of ASP3258-derived radioactivity. In vitro metabolic profiles were relatively similar among the species examined, suggesting that our findings in rats may help us to understand pharmacokinetics, efficacy and safety profiles in humans and other species.

  13. Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

    PubMed Central

    Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

    2016-01-01

    AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher

  14. Isomerization of Asp residues plays an important role in αA-crystallin dissociation.

    PubMed

    Takata, Takumi; Fujii, Noriko

    2016-03-01

    Aged cataract formation is caused by the accumulative precipitation of lens proteins incorporating diverse post-translational modifications. α-Crystallin, a major structural and functional lens protein, consists of a large polymeric structure that is dissociated and insolubilized with accumulative post-translational modifications. One such modification, isomerization of Asp, was recently identified in αB-crystallin monomers derived from aged lens. However, the distributions of Asp isomers in each lens fraction remain unknown. Here, α-crystallin fractions from aged lens were separated into heteropolymeric and monomeric forms to determine the Asp isomerization ratios in each fraction. Lens of four different ages were homogenized and centrifuged, and the soluble fraction was applied to size-exclusion chromatography. The heteropolymeric α-crystallin and monomeric crystallin fractions were obtained and concentrated. After trypsin digestion, each fraction was independently applied to liquid chromatography equipped with mass spectrometry to extract α-crystallin-derived peptides containing Asp isomers. The results showed that Asp58, Asp84 and Asp151 of αA-crystallin were highly isomerized in the monomeric fraction, but not isomerized to the same level in the heteropolymeric fraction. Each type of Asp isomerization increased in an age-dependent manner, was site-specific and was similar to previous results from lens water-insoluble fractions. These results imply that isomerization of Asp residues leads to dissociation of αA-crystallin from the heteropolymeric state and induces insolubilization in aged lens. Taken together, our findings suggest that isomerization of Asp might disrupt the higher order polymeric state of α-crystallin, resulting in decreased solubility and function, ultimately contributing to lens protein impairment and cataract formation with aging. PMID:26700637

  15. Isomerization of Asp residues plays an important role in αA-crystallin dissociation.

    PubMed

    Takata, Takumi; Fujii, Noriko

    2016-03-01

    Aged cataract formation is caused by the accumulative precipitation of lens proteins incorporating diverse post-translational modifications. α-Crystallin, a major structural and functional lens protein, consists of a large polymeric structure that is dissociated and insolubilized with accumulative post-translational modifications. One such modification, isomerization of Asp, was recently identified in αB-crystallin monomers derived from aged lens. However, the distributions of Asp isomers in each lens fraction remain unknown. Here, α-crystallin fractions from aged lens were separated into heteropolymeric and monomeric forms to determine the Asp isomerization ratios in each fraction. Lens of four different ages were homogenized and centrifuged, and the soluble fraction was applied to size-exclusion chromatography. The heteropolymeric α-crystallin and monomeric crystallin fractions were obtained and concentrated. After trypsin digestion, each fraction was independently applied to liquid chromatography equipped with mass spectrometry to extract α-crystallin-derived peptides containing Asp isomers. The results showed that Asp58, Asp84 and Asp151 of αA-crystallin were highly isomerized in the monomeric fraction, but not isomerized to the same level in the heteropolymeric fraction. Each type of Asp isomerization increased in an age-dependent manner, was site-specific and was similar to previous results from lens water-insoluble fractions. These results imply that isomerization of Asp residues leads to dissociation of αA-crystallin from the heteropolymeric state and induces insolubilization in aged lens. Taken together, our findings suggest that isomerization of Asp might disrupt the higher order polymeric state of α-crystallin, resulting in decreased solubility and function, ultimately contributing to lens protein impairment and cataract formation with aging.

  16. 42 CFR 414.806 - Penalties associated with the failure to submit timely and accurate ASP data.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... timely and accurate ASP data. 414.806 Section 414.806 Public Health CENTERS FOR MEDICARE & MEDICAID... Penalties associated with the failure to submit timely and accurate ASP data. Section 1847A(d)(4) specifies the penalties associated with misrepresentations associated with ASP data. If the Secretary...

  17. 42 CFR 414.806 - Penalties associated with the failure to submit timely and accurate ASP data.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... timely and accurate ASP data. 414.806 Section 414.806 Public Health CENTERS FOR MEDICARE & MEDICAID... associated with the failure to submit timely and accurate ASP data. Section 1847A(d)(4) specifies the penalties associated with misrepresentations associated with ASP data. If the Secretary determines that...

  18. 42 CFR 414.806 - Penalties associated with the failure to submit timely and accurate ASP data.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... timely and accurate ASP data. 414.806 Section 414.806 Public Health CENTERS FOR MEDICARE & MEDICAID... Penalties associated with the failure to submit timely and accurate ASP data. Section 1847A(d)(4) specifies the penalties associated with misrepresentations associated with ASP data. If the Secretary...

  19. 42 CFR 414.806 - Penalties associated with the failure to submit timely and accurate ASP data.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... timely and accurate ASP data. 414.806 Section 414.806 Public Health CENTERS FOR MEDICARE & MEDICAID... associated with the failure to submit timely and accurate ASP data. Section 1847A(d)(4) specifies the penalties associated with misrepresentations associated with ASP data. If the Secretary determines that...

  20. 42 CFR 414.806 - Penalties associated with the failure to submit timely and accurate ASP data.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... timely and accurate ASP data. 414.806 Section 414.806 Public Health CENTERS FOR MEDICARE & MEDICAID... Penalties associated with the failure to submit timely and accurate ASP data. Section 1847A(d)(4) specifies the penalties associated with misrepresentations associated with ASP data. If the Secretary...

  1. Utility and safety of procalcitonin in an antimicrobial stewardship program (ASP) in patients with malignancies.

    PubMed

    Liew, Y X; Lee, W; Cai, Y Y; Teo, J; Tang, S S-L; Ong, R W-Q; Lim, C L-L; Lingegowda, P B; Kwa, A L-H; Chlebicki, M P

    2012-11-01

    As data on procalcitonin utility in antibiotics discontinuation [under an antimicrobial stewardship program (ASP)] in patients with malignancies are lacking, we aimed to evaluate the utility of procalcitonin in an ASP in patients with malignancies. We conducted a retrospective review of the ASP database of all patients with malignancies in whom at least one procalcitonin level was taken and our ASP had recommended changes in carbapenem regimen, from January to December 2011. We compared clinical outcomes between two groups of patients: patients whose physicians accepted and those whose physicians rejected ASP interventions. There were 749 carbapenem cases reviewed. Ninety-nine were suggested to either de-escalate, discontinue antibiotics, or narrow the spectrum of empiric treatment, based on procalcitonin trends. While there was no statistical difference in the mortality within 30 days post-ASP intervention (accepted: 8/65 patients vs. rejected: 9/34 patients; p = 0.076), the median duration of carbapenem therapy was significantly shorter (5 vs. 7 days; p = 0.002). Procalcitonin use safely facilitates decisions on antibiotics discontinuation and de-escalation in patients with malignancies in the ASP.

  2. Loss of ASP but not ROPN1 reduces mammalian ciliary motility.

    PubMed

    Fiedler, Sarah E; Sisson, Joseph H; Wyatt, Todd A; Pavlik, Jacqueline A; Gambling, Todd M; Carson, Johnny L; Carr, Daniel W

    2012-01-01

    Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins [AKAP-associated sperm protein (ASP), ropporin (ROPN1), sperm protein 17 (SP17) and calcium binding tyrosine-(Y)-phosphorylation regulated protein (CABYR)] share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease.

  3. Loss of ASP but not ROPN1 reduces mammalian ciliary motility

    PubMed Central

    Fiedler, Sarah E.; Sisson, Joseph H.; Wyatt, Todd A.; Pavlik, Jacqueline A.; Gambling, Todd M.; Carson, Johnny L.; Carr, Daniel W.

    2011-01-01

    Protein kinase A (PKA) signaling is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ASP, ROPN1, SP17, and CABYR) share this highly conserved RII dimerization/docking (R2D2) domain. ASP and ROPN1 are 41% identical in sequence, interact with a variety of AKAPs in a manner similar to PKA, and are expressed in ciliated and flagellated human cells. To test the hypothesis that these proteins regulate motility, we developed mutant mouse lines lacking ASP or ROPN1. Both mutant lines produced normal numbers of cilia with intact ciliary ultrastructure. Lack of ROPN1 had no effect on ciliary motility. However, the beat frequency of cilia from mice lacking ASP is significantly slower than wild type, indicating that ASP signaling may regulate ciliary motility. This is the first demonstration of in vivo function for ASP. Similar localization of ASP in mice and humans indicates that these findings may translate to human physiology, and that these mice will be an excellent model for future studies related to the pathogenesis of human disease. PMID:22021175

  4. Possible mechanism of structural transformations induced by StAsp-PSI in lipid membranes.

    PubMed

    Muñoz, Fernando; Palomares-Jerez, M Francisca; Daleo, Gustavo; Villalaín, José; Guevara, M Gabriela

    2014-01-01

    In the present work we have analyzed the effect of StAsp-PSI (plant-specific insert of potato aspartic protease) on the structural and thermotropic properties of the major phospholipid types of bacterial and animal cells. Results obtained suggest that StAsp-PSI induces a destabilization of the membrane bilayers, depending on the time of interaction between the protein and the bilayers, rather than on its concentration. This temporal delay would be consistent with a lateral diffusion of StAsp-PSI monomers to assemble into aggregates to form pores. Like with the results previously reported for the StAsp-PSI circular dichroism, data obtained here from IR spectroscopy show that there are slight changes in the StAsp-PSI secondary structure in the presence of lipid membranes; suggesting that these changes could be related with the StAsp-PSI self-association. Results obtained from steady-state fluorescence anisotropy and differential scanning calorimetry assays suggest that StAsp-PSI interacts with both uncharged and negatively charged phospholipids, modulates the phase polymorphic behavior of model membranes and partitions and buries differentially in the membrane depending on the presence of negatively charged phospholipids.

  5. A Novel Asp121Asn Mutation of Myelin Protein Zero Is Associated with Late-Onset Axonal Charcot-Marie-Tooth Disease, Hearing Loss and Pupil Abnormalities

    PubMed Central

    Duan, Xiaohui; Gu, Weihong; Hao, Ying; Wang, Renbin; Wen, Hong; Sun, Shaojie; Jiao, Jinsong; Fan, Dongsheng

    2016-01-01

    Myelin protein zero (MPZ) is a major component of compact myelin in peripheral nerves. Mutations in MPZ have been associated with different Charcot–Marie–Tooth disease (CMT) phenotypes (CMT1B, CMT2I/J, CMTDI), Dejerine–Sottas syndrome, and congenital hypomyelination neuropathy. Here, we report phenotypic variability in a four-generation Chinese family with the MPZ mutation Asp121Asn. Genetic testing was performed on nine family members and 200 controls. Clinical, electrophysiological and skeletal muscle MRI assessments were available for review in six family members. A novel heterozygous missense mutation, Asp121Asn, was observed in five affected members of the family. Unaffected relatives and 200 normal controls were without the mutation. Four of the affected members of the family displayed late-onset, predominantly axonal sensory and motor neuropathy, pupil abnormalities, and progressive sensorineural hearing loss. One young affected member presented with Argyll–Robertson pupils and diminished deep tendon reflexes in the lower limbs. The MPZ mutation Asp121Asn may be associated with late-onset axonal neuropathy, early onset hearing loss and pupil abnormalities. Our report expands the number and phenotypic spectrum of MPZ mutations. PMID:27774063

  6. Hormone and pharmaceutical regulation of ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cianflone, Katherine

    2010-04-01

    Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

  7. Angel-shaped phalango-epiphyseal dysplasia (ASPED): identification of a new genetic bone marker.

    PubMed

    Giedion, A; Prader, A; Fliegel, C; Krasikov, N; Langer, L; Poznanski, A

    1993-10-01

    We describe a "new" mild malformation of the phalanx, which we call the "angel-shaped phalanx" (ASP) because of its resemblance to the little angels used for the decoration of Christmas trees. A particular middle phalangeal type of ASPs is found in a distinct variety of multiple epiphyseal dysplasia with marked retardation of bone age and severe coxarthrosis in adult life, previously reported as "hereditary peripheral dysostosis" [Bachman, 1967: Proc R Soc Med 60:21-22; Giedion, 1969: Fortschr Rontgenstr 110:507-524]. However, these authors overlooked the unique configuration of the middle phalanges. We renamed the condition "angel-shaped phalango-epiphyseal dysplasia (ASPED)", which may be transmitted in an autosomal-dominant manner. Six new patients are added, bringing the total to nine patients (two families and two isolated patients). ASPs were seen in five of six children. The ASPs grew into inconspicuous brachydactyly after physeal closure (3/3). The most important additional radiological finding is late and dysplastic development of both femoral heads (5/5), leading to Perthes-like and osteoarthritic changes and severe hip pain in the early thirties (2/2 adults, having reached this age). The marked retardation of carpal bone age may lead to unnecessary clinical evaluation for endocrine disorders. Less frequent clinical manifestations of ASPED are hyperextensibility of the interphalangeal joints (7/9) and hypodontia (4/7). Other types of ASPs are observed in brachyphalangy type C, spondylo-megepiphyseal-metaphyseal dysplasia, and other conditions. The concept of mild bone abnormalities as specific markers for genetic disease, as with cone-shaped epiphyses and now evident in ASPED, may also be useful for ASPs in general.

  8. An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.

    PubMed

    Cosgrove, M S; Gover, S; Naylor, C E; Vandeputte-Rutten, L; Adams, M J; Levy, H R

    2000-12-12

    The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.

  9. Mechanism of Asp24 Upregulation in Brucella abortus Rough Mutant with a Disrupted O-Antigen Export System and Effect of Asp24 in Bacterial Intracellular Survival

    PubMed Central

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Ding, Chan; Wang, Shaohui; Peng, Daxin

    2014-01-01

    We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants. PMID:24752516

  10. Mechanism of Asp24 upregulation in Brucella abortus rough mutant with a disrupted O-antigen export system and effect of Asp24 in bacterial intracellular survival.

    PubMed

    Tian, Mingxing; Qu, Jing; Han, Xiangan; Ding, Chan; Wang, Shaohui; Peng, Daxin; Yu, Shengqing

    2014-07-01

    We previously showed that Brucella abortus rough mutant strain 2308 ΔATP (called the ΔrfbE mutant in this study) exhibits reduced intracellular survival in RAW264.7 cells and attenuated persistence in BALB/c mice. In this study, we performed microarray analysis to detect genes with differential expression between the ΔrfbE mutant and wild-type strain S2308. Interestingly, acid shock protein 24 gene (asp24) expression was significantly upregulated in the ΔrfbE mutant compared to S2308, as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Further studies using additional strains indicated that the upregulation of asp24 occurred only in rough mutants with disrupted O-antigen export system components, including the ATP-binding protein gene rfbE (bab1_0542) and the permease gene rfbD (bab1_0543), while the ΔwboA rough mutant (which lacks an O-antigen synthesis-related glycosyltransferase) and the RB51 strain (a vaccine strain with the rough phenotype) showed no significant changes in asp24 expression compared to S2308. In addition, abolishing the intracellular O-antigen synthesis of the ΔrfbE mutant by deleting the wboA gene (thereby creating the ΔrfbE ΔwboA double-knockout strain) recovered asp24 expression. These results indicated that asp24 upregulation is associated with intracellular O-antigen synthesis and accumulation but not with the bacterial rough phenotype. Further studies indicated that asp24 upregulation in the ΔrfbE mutant was associated neither with bacterial adherence and invasion nor with cellular necrosis on RAW264.7 macrophages. However, proper expression of the asp24 gene favors intracellular survival of Brucella in RAW264.7 cells and HeLa cells during an infection. This study reveals a novel mechanism for asp24 upregulation in B. abortus mutants.

  11. Array Simulations Platform (ASP) predicts NASA Data Link Module (NDLM) performance

    NASA Technical Reports Server (NTRS)

    Snook, Allen David

    1993-01-01

    Through a variety of imbedded theoretical and actual antenna patterns, the array simulation platform (ASP) enhanced analysis of the array antenna pattern effects for the KTx (Ku-Band Transmit) service of the NDLM (NASA Data Link Module). The ASP utilizes internally stored models of the NDLM antennas and can develop the overall pattern of antenna arrays through common array calculation techniques. ASP expertly assisted in the diagnosing of element phase shifter errors during KTx testing and was able to accurately predict the overall array pattern from combinations of the four internally held element patterns. This paper provides an overview of the use of the ASP software in the solving of array mis-phasing problems.

  12. D-Asp: a new player in reproductive endocrinology of the amphibian Rana esculenta.

    PubMed

    Raucci, Franca; Di Fiore, Maria Maddalena

    2011-11-01

    We investigated the involvement of D-Aspartic acid (D-Asp) on ovarian and testicular morphology of the green frog, Rana esculenta, and its effect on the testosterone production. The study has been performed throughout the reproductive cycle. In both ovary and testis a substantial amount of D-Asp is endogenously present and its concentration varies as function of reproduction. In the frog, D-Asp content is differently correlated with gonadal and plasmatic levels of testosterone, depending on the sex. In fact, the amount of the D-Asp is inversely linked with that of the testosterone in the ovary, while this correlation directly matched in the testis. In vivo short-term experiments, consisting of a single intra-peritoneal injection of D-Asp (2.0 μmol/g body weight), demonstrated that the enantiomer is significantly accumulated by both the ovary and testis, reaching after 3 h the highest uptake and thereafter decreasing to baseline values within 24 h. Furthermore, D-Asp influences the synthesis and/or the release of testosterone, causing a decrease of its level in the female, and an increase in the male, respectively. In vivo long-term experiments, D-Asp, chronically administered to the frogs of both sexes, enhances the maturation of both gonads, determining in the oocytes an higher accumulation of carbohydrate yolk plates in the ooplasm, and stimulating the spermatogenesis in the testis. Taken altogether, our results show that D-Asp operates differently in female and male frog gonads, indicating that it has different targets in the reproductive machinery depending on the sex.

  13. Structure of Ostertagia ostertagi ASP-1: insights into disulfide-mediated cyclization and dimerization.

    PubMed

    Borloo, Jimmy; Geldhof, Peter; Peelaers, Iris; Van Meulder, Frederik; Ameloot, Paul; Callewaert, Nico; Vercruysse, Jozef; Claerebout, Edwin; Strelkov, Sergei V; Weeks, Stephen D

    2013-04-01

    The cysteine-rich secretory/antigen 5/pathogenesis-related 1 (CAP) protein superfamily is composed of a functionally diverse group of members that are found in both eukaryotes and prokaryotes. The excretome/secretome of numerous helminths (parasitic nematodes) contains abundant amounts of CAP members termed activation-associated secreted proteins (ASPs). Although ASPs are necessary for the parasitic life cycle in the host, the current lack of structural and functional information limits both understanding of their actual role in host-parasite interactions and the development of new routes in controlling parasitic infections and diseases. Alleviating this knowledge gap, a 1.85 Å resolution structure of recombinantly produced Oo-ASP-1 from Ostertagia ostertagi, which is one of the most prevalent gastrointestinal parasites in cattle worldwide, was solved. Overall, Oo-ASP-1 displays the common hallmark architecture shared by all CAP-superfamily members, including the N-terminal CAP and C-terminal cysteine-rich domains, but it also reveals a number of highly peculiar features. In agreement with studies of the natively produced protein, the crystal structure shows that Oo-ASP-1 forms a stable dimer that has been found to be primarily maintained via an intermolecular disulfide bridge, hence the small interaction surface of only 306.8 Å(2). Moreover, unlike any other ASP described to date, an additional intramolecular disulfide bridge links the N- and C-termini of each monomer, thereby yielding a quasi-cyclic molecule. Taken together, the insights presented here form an initial step towards a better understanding of the actual biological role(s) that this ASP plays in host-parasite interactions. The structure is also essential to help to define the key regions of the protein suitable for development of ASP-based vaccines, which would enable the current issues surrounding anthelmintic resistance in the treatment of parasitic infections and diseases to be circumvented.

  14. Efficacy of the investigational echinocandin ASP9726 in a guinea pig model of invasive pulmonary aspergillosis.

    PubMed

    Wiederhold, Nathan P; Najvar, Laura K; Matsumoto, Satoru; Bocanegra, Rosie A; Herrera, Monica L; Wickes, Brian L; Kirkpatrick, William R; Patterson, Thomas F

    2015-05-01

    ASP9726 is an investigational echinocandin with in vitro activity against Aspergillus species. We evaluated the pharmacokinetics and efficacy of this agent in an established guinea pig model of invasive pulmonary aspergillosis. ASP9726 plasma concentrations were measured in guinea pigs administered either a single dose or multiple doses of this agent at 2.5, 5, and 10 mg/kg of body weight/day by subcutaneous injection. Immunosuppressed guinea pigs were inoculated with A. fumigatus AF293, and ASP9726 (2.5, 5, and 10 mg/kg/day), voriconazole (10 mg/kg by oral gavage twice daily), or caspofungin (3 mg/kg/day by intraperitoneal injection) was administered for 8 days. Changes in fungal burden were measured by enumerating CFU and by quantitative PCR of specimens from within the lungs, as well as by analysis of serum (1 → 3)-β-D-glucan and galactomannan. Lung histopathology was also evaluated. ASP9726 plasma concentrations increased in a dose-proportional manner, and the drug was well tolerated at each dose. Each dose of ASP9726, voriconazole, and caspofungin significantly reduced pulmonary fungal burden as measured by quantitative PCR and by determining (1 → 3)-β-D-glucan and galactomannan levels, but only voriconazole significantly reduced numbers of CFU. ASP9726 at 5 mg/kg also significantly improved survival. Histopathology demonstrated morphological changes in hyphae in animals exposed to ASP9726 and caspofungin, consistent with the activities of the echinocandins. These results suggest that ASP9726 may be efficacious for the treatment of invasive pulmonary aspergillosis. PMID:25753643

  15. Fibrinogen Seoul (FGG Ala341Asp): a novel mutation associated with hypodysfibrinogenemia.

    PubMed

    Song, Kyung Soon; Park, Noh Jin; Choi, Jong Rak; Doh, Hyun Joo; Chung, Kwang Hoe

    2006-07-01

    Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.

  16. Anti-diabetic activities of Acanthopanax senticosus polysaccharide (ASP) in combination with metformin.

    PubMed

    Fu, Jianfang; Fu, Jufang; Yuan, Jun; Zhang, Nanyan; Gao, Bin; Fu, Guoqiang; Tu, Yanyang; Zhang, Yongsheng

    2012-04-01

    Combination therapy had become very popular currently for the diabetes mellitus and its complications, because of long term unreasonable drug use and adverse reaction to human body. In this study, a polysaccharide (ASP) from the roots of Acanthopanax senticosus was evaluated as an adjuvant with metformin for antidiabetic therapy in alloxan-induced diabetic rats. The result identified ASP plus metformin had a more beneficial promotion for relieving the symptoms of diabetes and reversing liver and kidney damage to normal level than only metfomin administration to diabetic rats. The blood glucose, blood lipid (TC and TG), thiobarbituric acid reactive substances (TBARS), AST, ALT, ALP, total bilirubin, creatinine and urea levels in diabetic rats were decreased by combination of ASP and metformin. Furthermore, the body weight, liver glycogen formation, antioxidant substance (GSH) and antioxidant enzyme (SOD and GPX) levels increased evidently in diabetic mice treated with both ASP and metformin. In particular, sometimes ASP plus metformin could significantly reverse the pathophysiologic parameters of diabetic rats to normal level than only metformin administration. Therefore ASP could be developed to a new adjuvant combined with metformin for diabetes mellitus therapy in the future.

  17. Stability of Asp(B28) Insulin Exposed to Modified and Unmodified Polypropylene.

    PubMed

    Fristrup, Charlotte J; Jankova, Katja; Eskimergen, Rüya; Bukrinsky, Jens T; Hvilsted, Soren

    2015-01-01

    Polypropylene (PP) plates have been modified with two different hydrophilic polymeric materials, poly(N,N-dimethylacrylamide) (poly(DMAAm)) and poly(poly(ethylene glycol)methacrylate) (poly(PEGMA)) in order to reduce insulin adsorption when the plates were exposed to insulin aspart (Asp(B28) insulin). The influence of surface modification on the chemical and physical stability of Asp(B28) insulin was evaluated by two chromatographic methods, size exclusion chromatography (SEC) and reverse phase high pressure liquid chromatography (RP-HPLC) and the Thioflavin T assay. A clear difference in the stability of Asp(B28) insulin was observed between the three tested surfaces. PP coated with poly(DMAAm) resulted in a poor chemical stability and a significantly improved physical stability compared with unmodified PP. In addition to this a lower phenol concentration was observed for the poly(DMAAm) coating. The results from the poly(PEGMA) coating looked very promising with respect to the stability of Asp(B28) insulin in comparison with the data from unmodified PP and the poly(DMAAm) coating. Two hydrophilic coatings have been tested and surprisingly a difference in Asp(B28) insulin stability was observed. Therefore, Asp(B28) insulin adsorption and stability will be influenced by more than the hydrophilicity of the surface.

  18. An Asp7Gly substitution in PPARG is associated with decreased transcriptional activation activity.

    PubMed

    Hua, Liushuai; Wang, Jing; Li, Mingxun; Sun, Xiaomei; Zhang, Liangzhi; Lei, Chuzhao; Lan, Xianyong; Fang, Xingtang; Zhao, Xin; Chen, Hong

    2014-01-01

    As the master regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARG) is required for the accumulation of adipose tissue and hence contributes to obesity. A previous study showed that the substitution of +20A>G in PPARG changed the 7(th) amino acid from Asp to Gly, creating a mutant referred to as PPARG Asp7Gly. In this study, association analysis indicated that PPARG Asp7Gly was associated with lower body height, body weight and heart girth in cattle (P<0.05). Overexpression of PPARG in NIH3T3-L1 cells showed that the Asp7Gly substitution may cause a decrease in its adipogenic ability and the mRNA levels of CIDEC (cell death-inducing DFFA-like effector c) and aP2, which are all transcriptionally activated by PPARG during adipocyte differentiation. A dual-luciferase reporter assay was used to analyze the promoter activity of CIDEC. The results confirmed that the mutant PPARG exhibited weaker transcriptional activation activity than the wild type (P<0.05). These findings likely explain the associations between the Asp7Gly substitution and the body measurements. Additionally, the Asp7Gly mutation may be used in molecular marker assisted selection (MAS) of cattle breeding in the future. PMID:24466299

  19. A cyclic nucleotide-gated channel mutation associated with canine daylight blindness provides insight into a role for the S2 segment tri-Asp motif in channel biogenesis.

    PubMed

    Tanaka, Naoto; Delemotte, Lucie; Klein, Michael L; Komáromy, András M; Tanaka, Jacqueline C

    2014-01-01

    Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1-S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1-S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during

  20. Comparative studies on the discrepant fragmentation mechanisms of the GLy-Asp-Gly-Arg and Arg-Gly-Asp-Gly: evidence for the mobile proton model.

    PubMed

    2014-01-01

    The fragmentation mechanisms of singly protonated Gly-Asp-Gly-Arg (GDGRI and Arg-Gly-Asp-Gly (RGDGJ were investigated by mass spectrometry and theoretical methods. Both protonated molecules are fragmented mainly at the Asp-Gly amide bond C-terminal to Asp, as supported by quantum chemical calculations. Charge distributions of C and N atoms (Qc + QN) on the amide bonds were collected when the ionizing proton was fixed at different nitrogen atoms along the backbone for each peptide. Compared with the neutral molecules, the total charges of C and N atoms (Qc + QN] for the singly charged peptides tended to be negative when the proton was located at the backbone nitrogen atoms. A relatively larger value of QC + QN corresponds to a higher trend of fragmentation, which is consistent with the experimental relative abundances data that the predominant ions are y2 for [GDGR + H]+ and b3 for [RGDG + H]+. Also, the anhydride mechanism driven by the C-terminal COOH for [RGDG + H]+ was explored by a quantum-mechanical/molecular-mechanical method. Calculations indicate that the protonated peptide can be cleaved through an unusual charge-directed pathway by forming a salt bridge at the C-termini. The formation of the anhydride linkage is much more feasible since this process needs very little energy and is exother- mic, though the subsequent nucleophilic attack on the Asp carbonyl carbon is more difficult. The combined experimental and theoretical methods substantiate the mobile proton model, which opens a way to analyze quantitatively the discrepant fragmentation of dissociated peptides in peptide/protein identification.

  1. Distinct Roles of the Active-site Mg2+ Ligands, Asp882 and Asp705, of DNA Polymerase I (Klenow Fragment) during the Prechemistry Conformational Transitions*

    PubMed Central

    Bermek, Oya; Grindley, Nigel D. F.; Joyce, Catherine M.

    2011-01-01

    DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg2+ ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp705 and Asp882, of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp882; this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp882 position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp705 carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg2+ into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg2+ concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing. PMID:21084297

  2. Implementation of an ASP model offsite backup archive for clinical images utilizing Internet 2

    NASA Astrophysics Data System (ADS)

    Liu, Brent J.; Chao, Sander S.; Documet, Jorge; Lee, Jasper; Lee, Michael; Topic, Ian; Williams, Lanita

    2005-04-01

    With the development of PACS technology and an increasing demand by medical facilities to become filmless, there is a need for a fast and efficient method of providing data backup for disaster recovery and downtime scenarios. At the Image Processing Informatics Lab (IPI), an ASP Backup Archive was developed using a fault-tolerant server with a T1 connection to serve the PACS at the St. John's Health Center (SJHC) Santa Monica, California. The ASP archive server has been in clinical operation for more than 18 months, and its performance was presented at this SPIE Conference last year. This paper extends the ASP Backup Archive to serve the PACS at the USC Healthcare Consultation Center II (HCC2) utilizing an Internet2 connection. HCC2 is a new outpatient facility that recently opened in April 2004. The Internet2 connectivity between USC's HCC2 and IPI has been established for over one year. There are two novelties of the current ASP model: 1) Use of Internet2 for daily clinical operation, and 2) Modifying the existing backup archive to handle two sites in the ASP model. This paper presents the evaluation of the ASP Backup Archive based on the following two criteria: 1) Reliability and performance of the Internet2 connection between HCC2 and IPI using DICOM image transfer in a clinical environment, and 2) Ability of the ASP Fault-Tolerant backup archive to support two separate clinical PACS sites simultaneously. The performances of using T1 and Internet2 at the two different sites are also compared.

  3. The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II.

    PubMed

    Rujano, Maria A; Sanchez-Pulido, Luis; Pennetier, Carole; le Dez, Gaelle; Basto, Renata

    2013-11-01

    Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. Here, we characterized the function of the Drosophila ASPM orthologue, Asp, and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. We show that size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. We propose that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness.

  4. Substrate stereoselectivity of poly(Asp) hydrolase-1 capable of cleaving β-amide bonds as revealed by investigation of enzymatic hydrolysis of stereoisomeric β-tri(Asp)s.

    PubMed

    Hiraishi, Tomohiro; Abe, Hideki; Maeda, Mizuo

    2015-12-01

    We previously reported that poly(Asp) hydrolase-1 (PahZ1KP-2) from Pedobacter sp. KP-2 selectively, but not completely, cleaved the amide bonds between β-Asp units in thermally synthesized poly(Asp) (tPAA). In the present study, the enzymatic hydrolysis of stereoisomeric β-tri(Asp)s by PahZ1KP-2 was investigated to clarify the substrate stereoselectivity of PahZ1KP-2 in the hydrolysis of tPAA. The results suggest the following structural features of PahZ1KP-2 at its substrate binding site: (1) the active site contains four subsites (2, 1, -1, and -2), three of which need to be occupied by Asp units for cleavage to occur; (2) for the hydrolysis to proceed, subsite 1 should be occupied by an L-Asp unit, whereas the other three subsites may accept both L- and D-Asp units; (3) for the two central subsites between which cleavage occurs, the (L-Asp)-(D-Asp) sequence is the most favorable for cleavage.

  5. Sequence and structural analysis of the Asp-box motif and Asp-box beta-propellers; a widespread propeller-type characteristic of the Vps10 domain family and several glycoside hydrolase families

    PubMed Central

    Quistgaard, Esben M; Thirup, Søren S

    2009-01-01

    Background The Asp-box is a short sequence and structure motif that folds as a well-defined β-hairpin. It is present in different folds, but occurs most prominently as repeats in β-propellers. Asp-box β-propellers are known to be characteristically irregular and to occur in many medically important proteins, most of which are glycosidase enzymes, but they are otherwise not well characterized and are only rarely treated as a distinct β-propeller family. We have analyzed the sequence, structure, function and occurrence of the Asp-box and s-Asp-box -a related shorter variant, and provide a comprehensive classification and computational analysis of the Asp-box β-propeller family. Results We find that all conserved residues of the Asp-box support its structure, whereas the residues in variable positions are generally used for other purposes. The Asp-box clearly has a structural role in β-propellers and is highly unlikely to be involved in ligand binding. Sequence analysis of the Asp-box β-propeller family reveals it to be very widespread especially in bacteria and suggests a wide functional range. Disregarding the Asp-boxes, sequence conservation of the propeller blades is very low, but a distinct pattern of residues with specific properties have been identified. Interestingly, Asp-boxes are occasionally found very close to other propeller-associated repeats in extensive mixed-motif stretches, which strongly suggests the existence of a novel class of hybrid β-propellers. Structural analysis reveals that the top and bottom faces of Asp-box β-propellers have striking and consistently different loop properties; the bottom is structurally conserved whereas the top shows great structural variation. Interestingly, only the top face is used for functional purposes in known structures. A structural analysis of the 10-bladed β-propeller fold, which has so far only been observed in the Asp-box family, reveals that the inner strands of the blades are unusually far apart

  6. Lack of association between the APEX1 Asp148Glu polymorphism and sporadic amyotrophic lateral sclerosis.

    PubMed

    Coppedè, Fabio; Lo Gerfo, Annalisa; Carlesi, Cecilia; Piazza, Selina; Mancuso, Michelangelo; Pasquali, Livia; Murri, Luigi; Migliore, Lucia; Siciliano, Gabriele

    2010-02-01

    Impairments in DNA repair enzymes have been observed in amyotrophic lateral sclerosis (ALS) tissues, particularly in the activity of the apurinic/apyrimidinic endonuclease 1 (APEX1). Moreover, it was suggested that the common APEX1 Asp148Glu polymorphism might be associated with ALS risk. To further address this question we performed the present study aimed at evaluating the contribution of the APEX1 Asp148Glu polymorphism in sporadic ALS (sALS) risk and clinical presentation, including age and site of onset and disease progression. We screened 134 sALS Italian patients and 129 matched controls for the presence of the APEX1 Asp148Glu polymorphism. No difference in APEX1 Asp148Glu allele and genotype frequencies was found between the groups, nor was the polymorphism associated with age and site of onset or disease progression. Present results do not support a role for the APEX1 Asp148Glu polymorphism in sALS pathogenesis in the Italian population. PMID:18482781

  7. Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2.

    PubMed

    Mason, Lyndel; Tribolet, Leon; Simon, Anne; von Gnielinski, Natascha; Nienaber, Lisa; Taylor, Paul; Willis, Charlene; Jones, Malcolm K; Sternberg, Paul W; Gasser, Robin B; Loukas, Alex; Hofmann, Andreas

    2014-05-01

    Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.

  8. Use of Computer Decision Support in an Antimicrobial Stewardship Program (ASP)

    PubMed Central

    Olson, J.A.; Stenehjem, E.; Buckel, W.R.; Thorell, E.A.; Howe, S.; Wu, X.; Jones, P.S.; Lloyd, J.F.

    2015-01-01

    Summary Objective Document information needs, gaps within the current electronic applications and reports, and workflow interruptions requiring manual information searches that decreased the ability of our antimicrobial stewardship program (ASP) at Intermountain Healthcare (IH) to prospectively audit and provide feedback to clinicians to improve antimicrobial use. Methods A framework was used to provide access to patient information contained in the electronic medical record, the enterprise-wide data warehouse, the data-driven alert file and the enterprise-wide encounter file to generate alerts and reports via pagers, emails and through the Centers for Diseases and Control’s National Healthcare Surveillance Network. Results Four new applications were developed and used by ASPs at Intermountain Medical Center (IMC) and Primary Children’s Hospital (PCH) based on the design and input from the pharmacists and infectious diseases physicians and the new Center for Diseases Control and Prevention/National Healthcare Safety Network (NHSN) antibiotic utilization specifications. Data from IMC and PCH now show a general decrease in the use of drugs initially targeted by the ASP at both facilities. Conclusions To be effective, ASPs need an enormous amount of “timely” information. Members of the ASP at IH report these new applications help them improve antibiotic use by allowing efficient, timely review and effective prioritization of patients receiving antimicrobials in order to optimize patient care. PMID:25848418

  9. A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without Schiff base deprotonation.

    PubMed

    Sonar, S; Marti, T; Rath, P; Fischer, W; Coleman, M; Nilsson, A; Khorana, H G; Rothschild, K J

    1994-11-18

    Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.

  10. Familiality of Tourette Syndrome, Obsessive-Compulsive Disorder, and Attention-Deficit/Hyperactivity Disorder: Heritability Analysis in a Large Sib-Pair Sample

    ERIC Educational Resources Information Center

    Mathews, Carol A.; Grados, Marco A.

    2011-01-01

    Objective: Tourette syndrome (TS) is a neuropsychiatric disorder with a genetic component that is highly comorbid with obsessive-compulsive disorder (OCD) and attention deficit/hyperactivity disorder (ADHD). However, the genetic relations between these disorders have not been clearly elucidated. This study examined the familial relations among TS,…

  11. X-CHROMOSOME INACTIVATION PATTERNS IN MONOZYGOTIC TWINS AND SIB PAIRS DISCORDANT FOR NON-SYNDROMIC CLEFT LIP AND/OR PALATE

    PubMed Central

    Kimani, Jane W.; Shi, Min; Daack-Hirsch, Sandra; Christensen, Kaare; Moretti-Ferreira, Danilo; Marazita, Mary L.; Field, Leigh L.; Canady, John W.; Murray, Jeffrey C.

    2009-01-01

    Background Orofacial clefts are common birth defects with a complex etiology. While underlying mechanisms are still largely unknown, altered gender ratios for clefting phenotypes, evidence for linkage to the X chromosome and the occurrence of several X-linked clefting syndromes suggest that skewed X chromosome inactivation (XCI) may contribute to the etiology of orofacial clefting. We tested this hypothesis in a sample set of female monozygotic (MZ) twins and sister pairs discordant for clefting. Methods We determined XCI in peripheral blood lymphocyte DNA using a methylation based androgen receptor gene assay. We measured skewing of XCI as the deviation in XCI pattern from a 50:50 ratio and used a paired t-test to compare the degree of skewing in cases and their unaffected sisters. Results Our analysis revealed no significant difference in the degree of skewing between twin pairs (P=0.3). However, significant differences were observed in the sister pairs (P=0.02), with the cleft lip with cleft palate (CL+P) group showing the most significant result (P=0.01). Results from the cleft lip only (P=0.79) and cleft palate only (P=0.75) groups were not significant. Conclusions We did not find evidence for involvement of skewed XCI in the discordance for clefting in our sample of female MZ twins. However, results from the paired sister study suggest that skewed XCI may be important in orofacial clefting, particularly CL+P. PMID:18000982

  12. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

    PubMed

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

    2016-01-01

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target. PMID:27624005

  13. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus

    PubMed Central

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B.; Bzymek, Krzysztof P.; Williams, John C.; Brakhage, Axel A.; Kalkum, Markus

    2016-01-01

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3’s peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target. PMID:27624005

  14. The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

    PubMed

    Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

    2016-09-14

    Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

  15. Cloning and characteristic analysis of a novel aspartic protease gene Asp55 from Trichoderma asperellum ACCC30536.

    PubMed

    Dou, Kai; Wang, Zhiying; Zhang, Rongshu; Wang, Na; Fan, Haijuan; Diao, Guiping; Liu, Zhihua

    2014-12-01

    Proteases secreted by fungi belonging to the genus Trichoderma play important roles in biocontrol. In this study, the coding sequence and promoter region of the novel aspartic protease gene Asp55 were cloned from strain Trichoderma asperellum ACCC30536. Many cis-elements involved in phytopathogenic and environmental stress responses were identified in the Asp55 promoter region and may be recognized by MYB or WRKY transcription factors. The expression pattern of Asp55 under eight culture conditions was investigated by RT-qPCR. The expression level of Asp55 was up-regulated by poplar stem powder, Alternaria alternata cell wall fragments and A. alternata fermentation liquid, while it was down-regulated by carbon and nitrogen source starvation, and by powdered poplar leaves and roots. Additionally, the expression patterns of 15 genes encoding MYB transcription factors (Myb1 to Myb15) were also analyzed by RT-qPCR. Myb2 showed the most similar expression pattern with Asp55. The cDNA of Asp55 was expressed in Escherichia coli BL21, and recombinant ASP55 (rASP55) was purified. The purified rASP55 was evaluated for enzymatic activity and showed inhibitory effect on phytopathogenic A. alternata.

  16. Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin.

    PubMed

    Zhang, Yanfang; Liu, Yu; Guo, Hui; Jiang, Wei; Dong, Ping; Liang, Xingguo

    2016-07-01

    Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity. PMID:27233129

  17. Introduction of the ASP3D Computer Program for Unsteady Aerodynamic and Aeroelastic Analyses

    NASA Technical Reports Server (NTRS)

    Batina, John T.

    2005-01-01

    A new computer program has been developed called ASP3D (Advanced Small Perturbation 3D), which solves the small perturbation potential flow equation in an advanced form including mass-consistent surface and trailing wake boundary conditions, and entropy, vorticity, and viscous effects. The purpose of the program is for unsteady aerodynamic and aeroelastic analyses, especially in the nonlinear transonic flight regime. The program exploits the simplicity of stationary Cartesian meshes with the movement or deformation of the configuration under consideration incorporated into the solution algorithm through a planar surface boundary condition. The new ASP3D code is the result of a decade of developmental work on improvements to the small perturbation formulation, performed while the author was employed as a Senior Research Scientist in the Configuration Aerodynamics Branch at the NASA Langley Research Center. The ASP3D code is a significant improvement to the state-of-the-art for transonic aeroelastic analyses over the CAP-TSD code (Computational Aeroelasticity Program Transonic Small Disturbance), which was developed principally by the author in the mid-1980s. The author is in a unique position as the developer of both computer programs to compare, contrast, and ultimately make conclusions regarding the underlying formulations and utility of each code. The paper describes the salient features of the ASP3D code including the rationale for improvements in comparison with CAP-TSD. Numerous results are presented to demonstrate the ASP3D capability. The general conclusion is that the new ASP3D capability is superior to the older CAP-TSD code because of the myriad improvements developed and incorporated.

  18. Electrospun PLA: PCL composites embedded with unmodified and 3-aminopropyltriethoxysilane (ASP) modified halloysite nanotubes (HNT)

    NASA Astrophysics Data System (ADS)

    Haroosh, Hazim J.; Dong, Yu; Chaudhary, Deeptangshu S.; Ingram, Gordon D.; Yusa, Shin-ichi

    2013-02-01

    Electrospinning is a simple and versatile fiber synthesis technique in which a high-voltage electric field is applied to a stream of polymer melt or polymer solution, resulting in the formation of continuous micro/nanofibers. Halloysite nanotubes (HNT) have been found to achieve improved structural and mechanical properties when embedded into various polymer matrices. This research work focuses on blending poly( ɛ-caprolactone) (PCL) (9 and 15 wt%/v) and poly(lactic acid) (PLA) (fixed at 8 wt%/v) solutions with HNT at two different concentrations 1 and 2 wt%/v. Both unmodified HNT and HNT modified with 3-aminopropyltriethoxysilane (ASP) were utilized in this study. Fiber properties have been shown to be strongly related to the solution viscosity and electrical conductivity. The addition of HNT increased the solution viscosity, thus resulting in the production of uniform fibers. For both PCL concentrations, the average fiber diameter increased with the increasing of HNT concentration. The average fiber diameters with HNT-ASP were reduced considerably in comparison to those with unmodified HNT when using 15 wt%/v PCL. Slightly better dispersion was obtained for PLA: PCL composites embedded with HNT-ASP compared to unmodified HNT. Furthermore, the addition of HNT-ASP to the polymeric blends resulted in a moderate decrease in the degree of crystallinity, as well as slight reductions of glass transition temperature of PCL, the crystallization temperature and melting temperature of PLA within composite materials. The infrared spectra of composites confirmed the successful embedding of HNT-ASP into PLA: PCL nanofibers relative to unmodified HNT due to the premodification using ASP to reduce the agglomeration behavior. This study provides a new material system that could be potentially used in drug delivery, and may facilitate good control of the drug release process.

  19. Nonlinear analysis and performance evaluation of the Annular Suspension and Pointing System (ASPS)

    NASA Technical Reports Server (NTRS)

    Joshi, S. M.

    1978-01-01

    The Annular Suspension and Pointing System (ASPS) can provide high accurate fine pointing for a variety of solar-, stellar-, and Earth-viewing scientific instruments during space shuttle orbital missions. In this report, a detailed nonlinear mathematical model is developed for the ASPS/Space Shuttle system. The equations are augmented with nonlinear models of components such as magnetic actuators and gimbal torquers. Control systems and payload attitude state estimators are designed in order to obtain satisfactory pointing performance, and statistical pointing performance is predicted in the presence of measurement noise and disturbances.

  20. ASPS performance with large payloads onboard the Shuttle Orbiter. [Annular Suspension and Pointing System

    NASA Technical Reports Server (NTRS)

    Keckler, C. R.

    1980-01-01

    A high fidelity digital computer simulation was used to establish the viability of the Annular Suspension and Pointing System (ASPS) for satisfying the pointing and stability requirements of facility class payloads, such as the Solar Optical Telescope, when subjected to the Orbiter disturbance environment. The ASPS and its payload were subjected to disturbances resulting from crew motions in the Orbiter aft flight deck and VRCS thruster firings. Worst case pointing errors of 0.005 arc seconds were experienced under the disturbance environment simulated; this is well within the 0.08 arc seconds requirement specified by the payload.

  1. Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

    PubMed

    Maeda, Hiroki; Takata, Takumi; Fujii, Norihiko; Sakaue, Hiroaki; Nirasawa, Satoru; Takahashi, Saori; Sasaki, Hiroshi; Fujii, Noriko

    2015-01-01

    Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lβ, Dβ, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lβ-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.

  2. Pharmacokinetics and Pharmacodynamics of ASP2151, a Helicase-Primase Inhibitor, in a Murine Model of Herpes Simplex Virus Infection

    PubMed Central

    Katsumata, Kiyomitsu; Chono, Koji; Kato, Kota; Ohtsu, Yoshiaki; Takakura, Shoji; Kontani, Toru

    2013-01-01

    ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus. Here, to determine and analyze the correlation between the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of ASP2151, we examined the PD profile of ASP2151 using in vitro plaque reduction assay and a murine model of HSV-1 infection. ASP2151 inhibited the in vitro replication of HSV-1 with a mean 50% effective concentration (EC50) of 14 ng/ml. In the cutaneously HSV-1-infected mouse model, ASP2151 dose dependently suppressed intradermal HSV-1 growth, with the effect reaching a plateau at a dose of 30 mg/kg of body weight/day. The dose fractionation study showed that intradermal HSV-1 titers were below the detection limit in mice treated with ASP2151 at 100 mg/kg/day divided into two daily doses and at 30 or 100 mg/kg/day divided into three daily doses. The intradermal HSV-1 titer correlated with the maximum concentration of drug in serum (Cmax), the area under the concentration-time curve over 24 h (AUC24h), and the time during which the concentration of ASP2151 in plasma was above 100 ng/ml (T>100). The continuous infusion of ASP2151 effectively decreased intradermal HSV-1 titers below the limit of detection in mice in which the ASP2151 concentration in plasma reached 79 to 145 ng/ml. Our findings suggest that the antiviral efficacy of ASP2151 is most closely associated with the PK parameter T>100 in HSV-1-infected mice. Based on these results, we propose that a plasma ASP2151 concentration exceeding 100 ng/ml for 21 to 24 h per day provides the maximum efficacy in HSV-1-infected mice. PMID:23274658

  3. Effect of ASP2151, a herpesvirus helicase-primase inhibitor, in a guinea pig model of genital herpes.

    PubMed

    Katsumata, Kiyomitsu; Chono, Koji; Sudo, Kenji; Shimizu, Yasuaki; Kontani, Toru; Suzuki, Hiroshi

    2011-08-25

    ASP2151 is a herpesvirus helicase-primase inhibitor with antiviral activity against varicella zoster virus and herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here, we examined the potency and efficacy of ASP2151 against HSV in vitro and in vivo. We found that ASP2151 was more potent in inhibiting the replication of HSV-1 and HSV-2 in Vero cells in the plaque reduction assay and had greater anti-HSV activity in a guinea pig model of genital herpes than did acyclovir and valacyclovir (VACV), respectively. Oral ASP2151 given from the day of infection reduced peak and overall disease scores in a dose-dependent manner, resulting in complete prevention of symptoms at the dose of 30 mg/kg. The 50% effective dose (ED(50)) values for ASP2151 and VACV were 0.37 and 68 mg/kg, respectively, indicating that ASP2151 was 184-fold more potent than VACV. When ASP2151 was administered after the onset of symptoms, the disease course of genital herpes was suppressed more effectively than by VACV, with a significant reduction in disease score observed one day after starting ASP2151 at 30 mg/kg, whereas the therapeutic effect of VACV was only evident three days after treatment at the highest dose tested (300 mg/kg). This indicated that ASP2151 possesses a faster onset of action and wider therapeutic time window than VACV. Further, virus shedding from the genital mucosa was significantly reduced with ASP2151 at 10 and 30 mg/kg but not with VACV, even at 300 mg/kg. Taken together, our present findings demonstrated the superior potency and efficacy of ASP2151 against HSV.

  4. Pharmacokinetics and pharmacodynamics of ASP2151, a helicase-primase inhibitor, in a murine model of herpes simplex virus infection.

    PubMed

    Katsumata, Kiyomitsu; Chono, Koji; Kato, Kota; Ohtsu, Yoshiaki; Takakura, Shoji; Kontani, Toru; Suzuki, Hiroshi

    2013-03-01

    ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus. Here, to determine and analyze the correlation between the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of ASP2151, we examined the PD profile of ASP2151 using in vitro plaque reduction assay and a murine model of HSV-1 infection. ASP2151 inhibited the in vitro replication of HSV-1 with a mean 50% effective concentration (EC(50)) of 14 ng/ml. In the cutaneously HSV-1-infected mouse model, ASP2151 dose dependently suppressed intradermal HSV-1 growth, with the effect reaching a plateau at a dose of 30 mg/kg of body weight/day. The dose fractionation study showed that intradermal HSV-1 titers were below the detection limit in mice treated with ASP2151 at 100 mg/kg/day divided into two daily doses and at 30 or 100 mg/kg/day divided into three daily doses. The intradermal HSV-1 titer correlated with the maximum concentration of drug in serum (C(max)), the area under the concentration-time curve over 24 h (AUC(24h)), and the time during which the concentration of ASP2151 in plasma was above 100 ng/ml (T(>100)). The continuous infusion of ASP2151 effectively decreased intradermal HSV-1 titers below the limit of detection in mice in which the ASP2151 concentration in plasma reached 79 to 145 ng/ml. Our findings suggest that the antiviral efficacy of ASP2151 is most closely associated with the PK parameter T(>100) in HSV-1-infected mice. Based on these results, we propose that a plasma ASP2151 concentration exceeding 100 ng/ml for 21 to 24 h per day provides the maximum efficacy in HSV-1-infected mice.

  5. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase.

    PubMed

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F

    2015-04-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase.

  6. Asp residues of βDELSEED-motif are required for peptide binding in the Escherichia coli ATP synthase

    PubMed Central

    Ahmad, Zulfiqar; Tayou, Junior; Laughlin, Thomas F.

    2015-01-01

    This study demonstrates the requirement of Asp-380 and Asp-386 in the βDELSEED-motif of E. coli ATP synthase for peptide binding and inhibition. We studied the inhibition profiles of wild-type and mutant E. coli ATP synthase in presence of c-terminal amide bound melittin and melittin related peptide. Melittin and melittin related peptide inhibited wild-type ATPase almost completely while only partial inhibition was observed in single mutations with replacement of Asp to Ala, Gln, or Arg. Additionally, very little or no inhibition occurred among double mutants βD380A/βD386A, βD380Q/βD386Q, or βD380R/βD386R signifying that removal of one Asp residue allows limited peptide binding. Partial or substantial loss of oxidative phosphorylation among double mutants demonstrates the functional requirement of βD380 and βD386 Asp residues. Moreover, abrogation of wild-type E. coli cell growth and normal growth of mutant cells in presence of peptides provides strong evidence for the requirement of βDELSEED-motif Asp residues for peptide binding. It is concluded that while presence of one Asp residue may allow partial peptide binding, both Asp residues, βD380 and βD386, are essential for proper peptide binding and inhibition of ATP synthase. PMID:25603139

  7. A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

    PubMed

    Brock, Joseph S; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-01-26

    Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

  8. Determination of the protonation state of the Asp dyad: conventional molecular dynamics versus thermodynamic integration.

    PubMed

    Huang, Jinfeng; Zhu, Yali; Sun, Bin; Yao, Yuan; Liu, Junjun

    2016-03-01

    The protonation state of the Asp dyad is important as it can reveal enzymatic mechanisms, and the information this provides can be used in the development of drugs for proteins such as memapsin 2 (BACE-1), HIV-1 protease, and rennin. Conventional molecular dynamics (MD) simulations have been successfully used to determine the preferred protonation state of the Asp dyad. In the present work, we demonstrate that the results obtained from conventional MD simulations can be greatly influenced by the particular force field applied or the values used for control parameters. In principle, free-energy changes between possible protonation states can be used to determine the protonation state. We show that protonation state prediction by the thermodynamic integration (TI) method is insensitive to force field version or to the cutoff for calculating nonbonded interactions (a control parameter). In the present study, the protonation state of the Asp dyad predicted by TI calculations was the same regardless of the force field and cutoff value applied. Contrary to the intuition that conventional MD is more efficient, our results clearly show that the TI method is actually more efficient and more reliable for determining the protonation state of the Asp dyad.

  9. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase).

    PubMed

    Rice, Amy J; Lei, Hao; Santarsiero, Bernard D; Lee, Hyun; Johnson, Michael E

    2016-10-01

    Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.

  10. Horst Aspöck, encyclopedist and entomologist extraordinaire - a personal appreciation.

    PubMed

    Ohl, Michael

    2016-01-01

    The paper provides an overview of the life and work of Prof. Dr. Horst Aspöck, the doyen of neuropterology, on the occasion of his 75(th) birthday. It particularly emphasizes his outstanding contributions to the development of neuropterology since the 1960s.

  11. Horst Aspöck, encyclopedist and entomologist extraordinaire – a personal appreciation

    PubMed Central

    Ohl, Michael

    2016-01-01

    Abstract The paper provides an overview of the life and work of Prof. Dr. Horst Aspöck, the doyen of neuropterology, on the occasion of his 75th birthday. It particularly emphasizes his outstanding contributions to the development of neuropterology since the 1960s. PMID:26884700

  12. Key Implementation Considerations for Executing Evidence-Based Programs: Project Overview. ASPE Research Brief

    ERIC Educational Resources Information Center

    US Department of Health and Human Services, 2013

    2013-01-01

    In April 2011, the U.S. Department of Health and Human Services (HHS) Office of the Assistant Secretary for Planning and Evaluation (ASPE) hosted a Forum, Emphasizing Evidence-Based Programs for Children and Youth, to convene the nation's leading practitioners and researchers with experience using and evaluating an array of evidence-based…

  13. TLR4 Asp299Gly (rs4986790) polymorphism and coronary artery disease: a meta-analysis

    PubMed Central

    Chen, Rui; Gao, Ying; Cen, Wei

    2015-01-01

    Background. Previous studies have shown conflicting results on the association between toll-like receptor 4 (TLR4) Asp299Gly (rs4986790) polymorphism and coronary artery disease (CAD). The aim of this study was to evaluate the influence of TLR4 Asp299Gly polymorphism on CAD risk, CRP level and the number of stenotic coronary arteries, as well as to investigate whether G allele carriers would benefit more from statin treatment. Methods. PubMed, EMBASE, and CNKI databases were searched until May 2015. All the statistical tests were performed using R version 3.1.2. Odds ratio (OR) and 95% confidence interval (CI) were used to assess the association between TLR4 Asp299Gly polymorphism and CAD risk, the number of stenotic vessels, and the incidence of cardiovascular events according to statin-treated patients. Weighted mean difference (WMD) was calculated for the association between Asp299Gly and CRP level. Results. Overall, 12 case-control studies with 10,258 cases and 5,891 controls were included, and no association of TLR4Asp299Gly polymorphism with CAD was found (G allele vs. A allele: OR = 0.97, 95% CI [0.81–1.17], P = 0.75; AA vs. GG + AG: OR = 0.97, 95% CI [0.80–1.18], P = 0.76; GG vs. AG + AA: OR = 1.08, 95% CI [0.57–2.02], P = 0.82; AG vs. AA + GG: OR = 1.03, 95% CI [0.85–1.25], P = 0.74). Also, no association was noted between Asp299Gly and CRP level (WMD = −0.10, 95% CI [−0.62, 0.41], P = 0.69). Furthermore, no synergistic effect of statin and 299Gly was reported (Statin_AA vs. Statin_AG/GG: OR = 1.12, 95% CI [0.41–3.09], P = 0.82). Discussion. This meta-analysis suggests no association of TLR4 Asp299Gly polymorphism with CAD and CRP level. It is further indicated that the G allele carriers may not benefit more from statin treatment. Further studies should include large sample size and high-quality literature to understand this issue in depth. PMID:26644971

  14. Properties and reactivity patterns of AsP(3): an experimental and computational study of group 15 elemental molecules.

    PubMed

    Cossairt, Brandi M; Cummins, Christopher C

    2009-10-28

    Facile synthetic access to the isolable, thermally robust AsP(3) molecule has allowed for a thorough study of its physical properties and reaction chemistry with a variety of transition-metal and organic fragments. The electronic properties of AsP(3) in comparison with P(4) are revealed by DFT and atoms in molecules (AIM) approaches and are discussed in relation to the observed electrochemical profiles and the phosphorus NMR properties of the two molecules. An investigation of the nucleus independent chemical shifts revealed that AsP(3) retains spherical aromaticity. The thermodynamic properties of AsP(3) and P(4) are described. The reaction types explored in this study include the thermal decomposition of the AsP(3) tetrahedron to its elements, the synthesis and structural characterization of [(AsP(3))FeCp*(dppe)][BPh(4)] (dppe = 1,2-bis(diphenylphosphino)ethane), 1, selective single As-P bond cleavage reactions, including the synthesis and structural characterization of AsP(3)(P(N((i)Pr)(2))N(SiMe(3))(2))(2), 2, and activations of AsP(3) by reactive early transition-metal fragments including Nb(H)(eta(2)-(t)Bu(H)C horizontal lineNAr)(N[CH(2)(t)Bu]Ar)(2) and Mo(N[(t)Bu]Ar)(3) (Ar = 3,5-Me(2)C(6)H(3)). In the presence of reducing equivalents, AsP(3) was found to allow access to [Na][E(3)Nb(ODipp)(3)] (Dipp = 2,6-diisopropylphenyl) complexes (E = As or P) which themselves allow access to mixtures of As(n)P(4-n) (n = 1-4).

  15. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    PubMed

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  16. Conserved Asp-137 is important for both structure and regulatory functions of cardiac α-tropomyosin (α-TM) in a novel transgenic mouse model expressing α-TM-D137L.

    PubMed

    Yar, Sumeyye; Chowdhury, Shamim A K; Davis, Robert T; Kobayashi, Minae; Monasky, Michelle M; Rajan, Sudarsan; Wolska, Beata M; Gaponenko, Vadim; Kobayashi, Tomoyoshi; Wieczorek, David F; Solaro, R John

    2013-06-01

    α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca(2+) sensitivity and cardiomyocyte contractility without changes in intracellular Ca(2+) transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

  17. Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L*

    PubMed Central

    Yar, Sumeyye; Chowdhury, Shamim A. K.; Davis, Robert T.; Kobayashi, Minae; Monasky, Michelle M.; Rajan, Sudarsan; Wolska, Beata M.; Gaponenko, Vadim; Kobayashi, Tomoyoshi; Wieczorek, David F.; Solaro, R. John

    2013-01-01

    α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca2+ sensitivity and cardiomyocyte contractility without changes in intracellular Ca2+ transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts. PMID:23609439

  18. Two new G gamma chain variants: Hb F-Saint-Etienne [G gamma 79(EF3)Asp-->His] and Hb F-Lyon [G gamma 97(FG4)His-->Arg].

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Berger, Claire; Perier, Christian; Barro, Claire; Francina, Alain

    2008-01-01

    Two new fetal hemoglobin (Hb F) variants affecting the (G)gamma chain are reported: Hb F-Saint-Etienne [G gamma 79(EF3)Asp-->His] and Hb F-Lyon [G gamma 97(FG4)His-->Arg]. These new Hb variants were found during a neonatal screening for hemoglobinopathies but characterized a few months later by our reference laboratory. The corresponding mutations are located on the external part of the Hb molecule and seem to be clinically silent.

  19. Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

    PubMed

    Xia, D; Peng, B; Sesok, D A; Peng, I

    1993-01-01

    We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril. PMID:8287497

  20. Asp96 deprotonation and transmembrane alpha-helical structural changes in bacteriorhodopsin.

    PubMed

    Rothschild, K J; Marti, T; Sonar, S; He, Y W; Rath, P; Fischer, W; Khorana, H G

    1993-12-25

    The M-->N transition in the photocycle of bacteriorhodopsin involves the transfer of a proton from Asp96 to the retinylidene Schiff base, possibly through a network of hydrogen-bonded amino acid residues and water molecules (Rothschild, K. J., He, Y. W., Sonar, S., Marti, T., and Khorana, H. G. (1992) J. Biol. Chem. 267, 1615-1622). A conformational change of the protein backbone is also observed during this transition. In this work, we have investigated the effects of replacing the residue Thr46, which might be part of this chain, with an aspartic acid. Both Fourier transform infrared and resonance Raman spectroscopy show that the chromophore structure of this mutant (T46D) is normal. However, N formation is accelerated and N decay is significantly slowed compared to wild-type bacteriorhodopsin. This effect causes the N intermediate to accumulate under steady-state illumination thereby facilitating spectroscopic studies under normal pH conditions. Fourier transform infrared difference spectroscopy reveals that like native bacteriorhodopsin, N formation in T46D involves deprotonation of Asp96, reprotonation of the Schiff base, and a change in the backbone secondary structure. However, in contrast to bacteriorhodopsin, bands assigned to the C = O stretch mode of the carboxylic acid group of Asp96 are upshifted by 10 cm-1 reflecting a change in the Asp96 environment and a drop in its effective pKa throughout the photocycle. This change in the pKa can directly account for changes in the photocycle kinetics and indicates that Asp96 deprotonation/protonation are the rate limiting steps in the formation and decay of the N intermediate. By studying the effects of H/D exchange, evidence is found that the backbone structural changes involve transmembrane alpha-helices. It is proposed that these structural changes serve to modulate the local environment and protonation state of Asp96 during the photocycle and are also essential for formation of the proton conducting hydrogen

  1. Biodegradation of phenanthrene in bioaugmented microcosm by consortium ASP developed from coastal sediment of Alang-Sosiya ship breaking yard.

    PubMed

    Patel, Vilas; Patel, Janki; Madamwar, Datta

    2013-09-15

    A phenanthrene-degrading bacterial consortium (ASP) was developed using sediment from the Alang-Sosiya shipbreaking yard at Gujarat, India. 16S rRNA gene-based molecular analyses revealed that the bacterial consortium consisted of six bacterial strains: Bacillus sp. ASP1, Pseudomonas sp. ASP2, Stenotrophomonas maltophilia strain ASP3, Staphylococcus sp. ASP4, Geobacillus sp. ASP5 and Alcaligenes sp. ASP6. The consortium was able to degrade 300 ppm of phenanthrene and 1000 ppm of naphthalene within 120 h and 48 h, respectively. Tween 80 showed a positive effect on phenanthrene degradation. The consortium was able to consume maximum phenanthrene at the rate of 46 mg/h/l and degrade phenanthrene in the presence of other petroleum hydrocarbons. A microcosm study was conducted to test the consortium's bioremediation potential. Phenanthrene degradation increased from 61% to 94% in sediment bioaugmented with the consortium. Simultaneously, bacterial counts and dehydrogenase activities also increased in the bioaugmented sediment. These results suggest that microbial consortium bioaugmentation may be a promising technology for bioremediation.

  2. Differential analysis of D-{beta}-Asp-containing proteins found in normal and infrared irradiated rabbit lens

    SciTech Connect

    Takata, Takumi; Shimo-Oka, Tadashi; Kojima, Masami; Miki, Kunio; Fujii, Noriko . E-mail: nfujii@HL.rri.kyoto-u.ac.jp

    2006-05-26

    Although proteins are generally composed of L-{alpha}-amino acids, D-{beta}-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several D-{beta}-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for D-{beta}-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel D-{beta}-Asp-containing proteins from rabbit lens. The results indicate that {beta}-crystallin A3, {beta}-crystallin A4, {beta}-crystallin B1, {beta}-crystallin B2, {beta}-crystallin B3, {gamma}-crystallin C, {gamma}-crystallin D, and {lambda}-crystallin in rabbit lens contain D-{beta}-Asp residues. Furthermore, the occurrence of D-{beta}-Asp residues increases with infrared ray (IR) irradiation. Additionally, some D-{beta}-Asp-containing proteins only appear after IR irradiation. One such protein is the {alpha}-enolase, which shows homology to {tau}-crystallin.

  3. Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle.

    PubMed

    Ito, Ami; Goshima, Gohta

    2015-12-01

    Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. We show that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. We identified a critical domain in Asp exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functioned as a global spindle coalescence factor not limited to MT ends. We propose a revised molecular model for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.

  4. CD4+ T cells mediate the protective effect of the recombinant Asp f3-based anti-aspergillosis vaccine.

    PubMed

    Diaz-Arevalo, Diana; Bagramyan, Karine; Hong, Teresa B; Ito, James I; Kalkum, Markus

    2011-06-01

    The mortality and morbidity caused by invasive aspergillosis present a major obstacle to the successful treatment of blood cancers with hematopoietic cell transplants. Patients who receive hematopoietic cell transplants are usually immunosuppressed for extended periods, and infection with the ubiquitous mold Aspergillus fumigatus is responsible for most cases of aspergillosis. Previously, we demonstrated that vaccination with recombinant forms of the A. fumigatus protein Asp f3 protected cortisone acetate-immunosuppressed mice from experimentally induced pulmonary aspergillosis. Here, we investigated the vaccine's protective mechanism and evaluated in particular the roles of antibodies and T cells. After vaccination, Asp f3-specific preinfection IgG titers did not significantly differ between surviving and nonsurviving mice, and passive transfer of anti-Asp f3 antibodies did not protect immunosuppressed recipients from aspergillosis. We experimentally confirmed Asp f3's predicted peroxisomal localization in A. fumigatus hyphae. We found that fungal Asp f3 is inaccessible to antibodies, unless both cell walls and membranes have been permeabilized. Antibody-induced depletion of CD4+ T cells reduced the survival of recombinant Asp f3 (rAsp f3)-vaccinated mice to nonimmune levels, and transplantation of purified CD4+ T cells from rAsp f3-vaccinated mice into nonimmunized recipients transferred antifungal protection. In addition, residues 60 to 79 and 75 to 94 of Asp f3 contain epitopes that induce proliferation of T cells from vaccinated survivors. Vaccine-primed CD4+ T cells are not expected to clear the fungal pathogen directly; however, they may locally activate immunosuppressed phagocytes that elicit the antifungal effect.

  5. Design of the annular suspension and pointing system /ASPS/ through decoupling and pole placement. [for Space Shuttle

    NASA Technical Reports Server (NTRS)

    Kuo, B. C.; Lin, W. C. W.

    1980-01-01

    A decoupling and pole-placement technique has been developed for the Annular Suspension and Pointing System (ASPS) of the Space Shuttle which uses bandwidths as performance criteria. The dynamics of the continuous-data ASPS allows the three degrees of freedom to be totally decoupled by state feedback through constant gains, so that the bandwidth of each degree of freedom can be independently specified without interaction. Although it is found that the digital ASPS cannot be completely decoupled, the bandwidth requirements are satisfied by pole placement and a trial-and-error method based on approximate decoupling.

  6. Application of the ASP3D Computer Program to Unsteady Aerodynamic and Aeroelastic Analyses

    NASA Technical Reports Server (NTRS)

    Batina, John T.

    2006-01-01

    A new computer program has been developed called ASP3D (Advanced Small Perturbation - 3D), which solves the small perturbation potential flow equation in an advanced form including mass-consistent surface and trailing wake boundary conditions, and entropy, vorticity, and viscous effects. The purpose of the program is for unsteady aerodynamic and aeroelastic analyses, especially in the nonlinear transonic flight regime. The program exploits the simplicity of stationary Cartesian meshes with the movement or deformation of the configuration under consideration incorporated into the solution algorithm through a planar surface boundary condition. The paper presents unsteady aerodynamic and aeroelastic applications of ASP3D to assess the time dependent capability and demonstrate various features of the code.

  7. ASP (AntiSubmarine Penetrator) base plate redesign and explosive bolt test

    SciTech Connect

    Cole, J.K.; Wolfe, W.P.

    1988-10-01

    This report presents the results of a post-flight investigation of the Rocket Antisubmarine Penetrator (RAP) tests of the AntiSubmarine Penetrator (ASP). It focuses on the cause for the premature deployment of the on-board recovery system and the failure of the base pressure transducers. As a result of the investigation, the base plate of the ASP vehicle was modified to increase its structural stiffness. Also, an instrumented test was conducted to assess the environment that is created when the three explosive bolts are activated to separate the vehicle from the interstage adapter and the rocket booster. The results of this test are presented and discussed. 5 refs., 15 figs.

  8. Low-power and shutdown models for the accident sequence precursor (ASP) program

    SciTech Connect

    Sattison, M.B.; Thatcher, T.A.; Knudsen, J.K.

    1997-02-01

    The US Nuclear Regulatory Commission (NRC) has been using full-power. Level 1, limited-scope risk models for the Accident Sequence Precursor (ASP) program for over fifteen years. These models have evolved and matured over the years, as have probabilistic risk assessment (PRA) and computer technologies. Significant upgrading activities have been undertaken over the past three years, with involvement from the Offices of Nuclear Reactor Regulation (NRR), Analysis and Evaluation of Operational Data (AEOD), and Nuclear Regulatory Research (RES), and several national laboratories. Part of these activities was an RES-sponsored feasibility study investigating the ability to extend the ASP models to include contributors to core damage from events initiated with the reactor at low power or shutdown (LP/SD), both internal events and external events. This paper presents only the LP/SD internal event modeling efforts.

  9. Microstructure simulation of rapidly solidified ASP30 high-speed steel particles by gas atomization

    NASA Astrophysics Data System (ADS)

    Ma, Jie; Wang, Bo; Yang, Zhi-liang; Wu, Guang-xin; Zhang, Jie-yu; Zhao, Shun-li

    2016-03-01

    In this study, the microstructure evolution of rapidly solidified ASP30 high-speed steel particles was predicted using a simulation method based on the cellular automaton-finite element (CAFE) model. The dendritic growth kinetics, in view of the characteristics of ASP30 steel, were calculated and combined with macro heat transfer calculations by user-defined functions (UDFs) to simulate the microstructure of gas-atomized particles. The relationship among particle diameter, undercooling, and the convection heat transfer coefficient was also investigated to provide cooling conditions for simulations. The simulated results indicated that a columnar grain microstructure was observed in small particles, whereas an equiaxed microstructure was observed in large particles. In addition, the morphologies and microstructures of gas-atomized ASP30 steel particles were also investigated experimentally using scanning electron microscopy (SEM). The experimental results showed that four major types of microstructures were formed: dendritic, equiaxed, mixed, and multi-droplet microstructures. The simulated results and the available experimental data are in good agreement.

  10. Characterization and Evolutionary Implications of the Triad Asp-Xxx-Glu in Group II Phosphopantetheinyl Transferases

    PubMed Central

    Wang, Yue-Yue; Li, Yu-Dong; Liu, Jian-Bo; Ran, Xin-Xin; Guo, Yuan-Yang; Ren, Ni-Ni; Chen, Xin; Jiang, Hui; Li, Yong-Quan

    2014-01-01

    Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes. PMID:25036863

  11. A critical amino acid residue, asp446, in UDP-glucuronosyltransferase.

    PubMed Central

    Iwano, H; Yokota, H; Ohgiya, S; Yotumoto, N; Yuasa, A

    1997-01-01

    An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from YshA1337GG1384A had no transferase activity. Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme. PMID:9271076

  12. Characterization and evolutionary implications of the triad Asp-Xxx-Glu in group II phosphopantetheinyl transferases.

    PubMed

    Wang, Yue-Yue; Li, Yu-Dong; Liu, Jian-Bo; Ran, Xin-Xin; Guo, Yuan-Yang; Ren, Ni-Ni; Chen, Xin; Jiang, Hui; Li, Yong-Quan

    2014-01-01

    Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.

  13. Laboratory evaluation of the pointing stability of the ASPS Vernier System

    NASA Technical Reports Server (NTRS)

    1980-01-01

    The annular suspension and pointing system (ASPS) is an end-mount experiment pointing system designed for use in the space shuttle. The results of the ASPS Vernier System (AVS) pointing stability tests conducted in a laboratory environment are documented. A simulated zero-G suspension was used to support the test payload in the laboratory. The AVS and the suspension were modelled and incorporated into a simulation of the laboratory test. Error sources were identified and pointing stability sensitivities were determined via simulation. Statistical predictions of laboratory test performance were derived and compared to actual laboratory test results. The predicted mean pointing stability during simulated shuttle disturbances was 1.22 arc seconds; the actual mean laboratory test pointing stability was 1.36 arc seconds. The successful prediction of laboratory test results provides increased confidence in the analytical understanding of the AVS magnetic bearing technology and allows confident prediction of in-flight performance. Computer simulations of ASPS, operating in the shuttle disturbance environment, predict in-flight pointing stability errors less than 0.01 arc seconds.

  14. Ser67Asp and His68Asp substitutions in candida parapsilosis carbonyl reductase alter the coenzyme specificity and enantioselectivity of ketone reduction.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Sun, Ying; Zhang, Wenchi; Xiao, Rong

    2009-04-01

    A short-chain carbonyl reductase (SCR) from Candida parapsilosis catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (R)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the k(cat)/K(m) value when NADH and NADPH were used as the cofactors, respectively, but maintaining a k(cat) value essentially the same with respect to wild-type SCR. The ratio of K(d) (dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.

  15. Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate.

    PubMed

    Guccione, Edward; Leon-Kempis, Maria del Rocio; Pearson, Bruce M; Hitchin, Edward; Mulholland, Francis; van Diemen, Pauline M; Stevens, Mark P; Kelly, David J

    2008-07-01

    Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA-dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.

  16. The Aspergillus Genome Database (AspGD): recent developments in comprehensive multispecies curation, comparative genomics and community resources.

    PubMed

    Arnaud, Martha B; Cerqueira, Gustavo C; Inglis, Diane O; Skrzypek, Marek S; Binkley, Jonathan; Chibucos, Marcus C; Crabtree, Jonathan; Howarth, Clinton; Orvis, Joshua; Shah, Prachi; Wymore, Farrell; Binkley, Gail; Miyasato, Stuart R; Simison, Matt; Sherlock, Gavin; Wortman, Jennifer R

    2012-01-01

    The Aspergillus Genome Database (AspGD; http://www.aspgd.org) is a freely available, web-based resource for researchers studying fungi of the genus Aspergillus, which includes organisms of clinical, agricultural and industrial importance. AspGD curators have now completed comprehensive review of the entire published literature about Aspergillus nidulans and Aspergillus fumigatus, and this annotation is provided with streamlined, ortholog-based navigation of the multispecies information. AspGD facilitates comparative genomics by providing a full-featured genomics viewer, as well as matched and standardized sets of genomic information for the sequenced aspergilli. AspGD also provides resources to foster interaction and dissemination of community information and resources. We welcome and encourage feedback at aspergillus-curator@lists.stanford.edu.

  17. In Vitro Activity of ASP2397 against Aspergillus Isolates with or without Acquired Azole Resistance Mechanisms

    PubMed Central

    Jensen, Rasmus Hare; Cuenca-Estrella, Manuel

    2015-01-01

    ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50 values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus

  18. Plasma Asp13-Ki-ras oncoprotein expression in vinyl chloride monomer workers in Taiwan.

    PubMed

    Luo, J C; Liu, H T; Cheng, T J; Du, C L; Wang, J D

    1998-12-01

    Vinyl chloride (VC) workers are known to be at risk for development of liver angiosarcoma, a rare tumor. Previously, more than 80% of VC workers with liver angiosarcoma have been found to have an Asp-13 c-Ki-ras oncogene mutation, and more than 50% of VC-exposed workers without liver tumors were found to have Asp13-Ki-ras oncoprotein in their plasma. Some workers in Taiwan had also been exposed to VC, and some have contracted liver tumors. In this study, we used enhanced chemiluminescence Western blotting to detect Asp13-p21-Ki-ras in the sera of VC-exposed workers in Taiwan. There were 14 of 113 (12.4%) VC workers positive for the Asp13-Ki-ras oncoprotein in plasma, but 0 of 18 controls were positive. There were 10 of 69 (14.5%) plasma-positives among the more highly exposed (> 1000 ppm-months) workers and 4 of 48 (9.1%) plasma-positives among the lesser exposed (< or = 1000 ppm-months). Compared with the unexposed controls, the odds ratios (and 95% confidence intervals [CI]) for plasma-positivity were 4.11 (95% CI = 0.21, 80.4) in the lower-exposed workers and 6.53 (95% CI = 0.37, 116.9) in the higher-exposed workers, and there was a linear trend between exposure and plasma-positivity (P = 0.073). After adjusting for age and drinking status, the odds ratios (and 95% CIs) were 1.64 (95% CI = 0.17, 15.8), and 2.65 (95% CI = 0.42, 16.8), respectively, and there was a significant linear trend between exposure and plasma-positivity (P = 0.048). In summary, Asp13-Ki-ras oncoprotein can be found in the plasma of VC workers in Taiwan, and a significant dose-response relationship exists between plasma oncoprotein expression and VC exposure.

  19. In Vitro Activity of ASP2397 against Aspergillus Isolates with or without Acquired Azole Resistance Mechanisms.

    PubMed

    Arendrup, Maiken Cavling; Jensen, Rasmus Hare; Cuenca-Estrella, Manuel

    2016-01-01

    ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50 values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus

  20. In Vitro Activity of ASP2397 against Aspergillus Isolates with or without Acquired Azole Resistance Mechanisms.

    PubMed

    Arendrup, Maiken Cavling; Jensen, Rasmus Hare; Cuenca-Estrella, Manuel

    2015-11-09

    ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50 values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus

  1. Use of the air pouch model to investigate immune responses to a hookworm vaccine containing the Na-ASP-2 protein in rats.

    PubMed

    Mendez, S; D' Samuel, A; Antoine, A D; Ahn, S; Hotez, P

    2008-01-01

    Hookworms are gastrointestinal nematodes that affect approximately 600 million people in developing countries. Using the air pouch model, we have examined the effects of vaccination with the recombinant hookworm larval antigen Na-ASP-2 and the adjuvant Alhydrogel on the skin immune response to hookworms in Sprague Dawley rats. Following vaccination, rats were inoculated 100 Necator americanus L3 into the air pouch, and the exudates and cell infiltrates were collected from the pouch 24 h later. Larval inoculation induced leucocyte recruitment into the pouch. Exudates of rats vaccinated with Na-ASP-2 showed an increase of cytokines such as IL-4, IL-10, IFN-gamma and especially, IL-5, as well as IgG1 and IgG2 antibodies. The increased amount of antigen-presenting cells and cytokines in the pouch of vaccinated animals suggests that vaccination could potentially restrain this parasite to the inoculation site, avoiding its migration and establishment in the host. Moreover, the air pouch model could constitute an alternative to screen immune responses to L3 antigens.

  2. Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant, rOv-ASP-1.

    PubMed

    Jiang, Jiu; Fisher, Erin M; Concannon, Mark; Lustigman, Sara; Shen, Hao; Murasko, Donna M

    2016-02-10

    Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV+rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV+rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly.

  3. Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant, rOv-ASP-1.

    PubMed

    Jiang, Jiu; Fisher, Erin M; Concannon, Mark; Lustigman, Sara; Shen, Hao; Murasko, Donna M

    2016-02-10

    Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV+rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV+rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly. PMID:26795365

  4. Cloning, expression, and characterization of a milk-clotting aspartic protease gene (Po-Asp) from Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Chen, Liguo; Tan, Qi; Shang, Xiaodong; Ma, Aimin

    2014-02-01

    An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3' portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68-80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.

  5. Characterization of the 41kDa allergen Asp v 13, a subtilisin-like serine protease from Aspergillus versicolor.

    PubMed

    Shi, C; Miller, J D

    2011-09-01

    Aspergillus versicolor is common on moldy building materials. Asp v 13, the principal allergen is produced by strains collected from across Canada. In this paper, we report a 1833bp Asp v 13 open reading frame predicted to encode a protein of 403 amino acids in length with three introns. A BLAST search of Asp v 13, a phylogenic tree calculation and alignment with its homologous proteins from other species indicated that Asp v 13 is a secretory, subtilisin-like serine protease widely distributed in Aspergillus species. His-tagged Asp v 13 was over-expressed in Escherichia coli and purified using Ni-NTA columns with a yield of 1mg/L. Based on immuno binding assay of recombinant protein both antibodies developed against the natural protein, and human sera IgE, the recombinant protein was similar to the natural form. Six IgE- and seven IgG-binding epitopes were also identified with selected human sera along the entire amino acid sequence of Asp v 13. Most residues binding these epitopes are exposed on the surface and correspond to charged regions of the molecule.

  6. Susceptibility of herpes simplex virus isolated from genital herpes lesions to ASP2151, a novel helicase-primase inhibitor.

    PubMed

    Katsumata, Kiyomitsu; Weinberg, Adriana; Chono, Koji; Takakura, Shoji; Kontani, Toru; Suzuki, Hiroshi

    2012-07-01

    ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. To evaluate the anti-HSV activity of ASP2151, susceptibility testing was performed on viruses isolated from patients participating in a placebo- and valacyclovir-controlled proof-of-concept phase II study for recurrent genital herpes. A total of 156 HSV strains were isolated prior to the dosing of patients, and no preexisting variants with less susceptibility to ASP2151 or acyclovir (ACV) were detected. ASP2151 inhibited HSV-1 and HSV-2 replication with mean 50% effective concentrations (EC(50)s) of 0.043 and 0.069 μM, whereas ACV exhibited mean EC(50)s of 2.1 and 3.2 μM, respectively. Notably, the susceptibilities of HSV isolates to ASP2151 and ACV were not altered after dosing with the antiviral agents. Taken together, these results demonstrate that ASP2151 inhibits the replication of HSV clinical isolates more potently than ACV, and HSV resistant to this novel helicase-primase inhibitor as well as ACV may not easily emerge in short-term treatment for recurrent genital herpes patients.

  7. Susceptibility of Herpes Simplex Virus Isolated from Genital Herpes Lesions to ASP2151, a Novel Helicase-Primase Inhibitor

    PubMed Central

    Katsumata, Kiyomitsu; Weinberg, Adriana; Chono, Koji; Takakura, Shoji; Kontani, Toru

    2012-01-01

    ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. To evaluate the anti-HSV activity of ASP2151, susceptibility testing was performed on viruses isolated from patients participating in a placebo- and valacyclovir-controlled proof-of-concept phase II study for recurrent genital herpes. A total of 156 HSV strains were isolated prior to the dosing of patients, and no preexisting variants with less susceptibility to ASP2151 or acyclovir (ACV) were detected. ASP2151 inhibited HSV-1 and HSV-2 replication with mean 50% effective concentrations (EC50s) of 0.043 and 0.069 μM, whereas ACV exhibited mean EC50s of 2.1 and 3.2 μM, respectively. Notably, the susceptibilities of HSV isolates to ASP2151 and ACV were not altered after dosing with the antiviral agents. Taken together, these results demonstrate that ASP2151 inhibits the replication of HSV clinical isolates more potently than ACV, and HSV resistant to this novel helicase-primase inhibitor as well as ACV may not easily emerge in short-term treatment for recurrent genital herpes patients. PMID:22526302

  8. Effects of NOS3 Glu298Asp polymorphism on hemodynamic reactivity to stress: influences of ethnicity and obesity.

    PubMed

    Malhotra, Surender; Poole, Joseph; Davis, Harry; Dong, Yanbin; Pollock, Jennifer; Snieder, Harold; Treiber, Frank

    2004-12-01

    Studies on the associations between the nitric oxide synthase gene (NOS3) Glu298Asp polymorphism and hypertension status or blood pressure (BP) levels have had inconsistent results. Potential moderating influences of ethnicity, sex, and obesity on the effects of the NOS3 polymorphism have not been examined. We evaluated the influence of these factors on associations between the NOS3 polymorphism, nitric oxide metabolites (NOx), and hemodynamics at rest and during stress. Subjects were 235 African American (AA) and 262 European American (EA) young adults (18.5+/-2.6 years). Hemodynamic measurements and blood samples for NOx assays were taken before and after a competitive video game challenge. Glu298Asp polymorphism was detected by polymerase chain reaction-restriction enzyme digestion assay. A regression model was built using genotypes, ethnicity, sex, and obesity (body mass index >85th percentile) and their interactions controlling for age; 20.1% of AAs and 49.8% of EAs were carriers of the Asp allele. AAs, regardless of obesity status, exhibited high diastolic blood pressure (DBP) reactivity unless they were nonobese and noncarriers of the Asp allele. EAs exhibited lower DBP reactivity unless they were obese Asp allele carriers. AA nonobese carriers exhibited the greatest total peripheral resistance reactivity. Obese Asp allele carriers exhibited the greatest increases in cardiac output and the greatest decrease in NOx to the stressor. Results indicate the importance of examining impact of BP control-related genetic polymorphisms within the context of moderating factors such as adiposity and ethnicity.

  9. Human mass balance, metabolite profile and identification of metabolic enzymes of [¹⁴C]ASP015K, a novel oral janus kinase inhibitor.

    PubMed

    Oda, Kazuo; Cao, Ying J; Sawamoto, Taiji; Nakada, Naoyuki; Fisniku, Ogert; Nagasaka, Yasuhisa; Sohda, Kin-Ya

    2015-01-01

    1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms. PMID:25986538

  10. Human mass balance, metabolite profile and identification of metabolic enzymes of [¹⁴C]ASP015K, a novel oral janus kinase inhibitor.

    PubMed

    Oda, Kazuo; Cao, Ying J; Sawamoto, Taiji; Nakada, Naoyuki; Fisniku, Ogert; Nagasaka, Yasuhisa; Sohda, Kin-Ya

    2015-01-01

    1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.

  11. First results from ASP on resonance production in. gamma gamma. interactions

    SciTech Connect

    Roe, N.

    1988-05-01

    The reaction e/sup +/e/sup -//yields/e/sup +/e/sup -//gamma/sup *//gamma/sup *///yields/(e/sup +/e/sup -/)/eta/, with subsequent decay of the /eta/ into two photons, has been observed with the ASP detector at the PEP e/sup +/e/sup -/ storage ring at /radical/ s=29 GeV. A measurement of the radiative width of the /eta/ yields the preliminary result /Gamma/(/eta//yields//gamma//gamma/) = .489 /+-/ .009 /+-/ .055 keV. Evidence for the production of the /eta/' with decay into two photons has also been observed.

  12. Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.

    PubMed

    Barbagallo, Michael S; Birch, Katherine E; Deacon, Nicholas J; Mosse, Jennifer A

    2012-07-01

    The negative-sense asp open reading frame (ORF) positioned opposite to the human immunodeficiency virus type 1 (HIV-1) env gene encodes the 189 amino acid, membrane-associated ASP protein. Negative-sense transcription, regulated by long terminal repeat sequences, has been observed early in HIV-1 infection in vitro. All subtypes of HIV-1 were scanned to detect the negative-sense asp ORF and to identify potential regulatory sequences. A series of highly conserved upstream short open reading frames (sORFs) was identified. This potential control region from HIV-1(NL4-3), containing six sORFs, was cloned upstream of the reporter gene EGFP. Expression by transfection of HEK293 cells indicated that the introduction of this sORF region inhibits EGFP reporter expression; analysis of transcripts revealed no significant changes in levels of EGFP mRNA. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) further demonstrated that the upstream sORF region undergoes alternative splicing in vitro. The most abundant product is spliced to remove sORFs I to V, leaving only the in-frame sORF VI upstream of asp. Sequence analysis revealed the presence of typical splice donor- and acceptor-site motifs. Mutation of the highly conserved splice donor and acceptor sites modulates, but does not fully relieve, inhibition of EGFP production. The strong conservation of asp and its sORFs across all HIV-1 subtypes suggests that the asp gene product may have a role in the pathogenesis of HIV-1. Alternative splicing of the upstream sORF region provides a potential mechanism for controlling expression of the asp gene.

  13. Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676, and the effect of D-Asp and iso-Asp substitution at Asp₆₇₂ residue.

    PubMed

    Shanmugam, Ganesh; Polavarapu, Prasad L; Láng, Emma; Majer, Zsuzsa

    2012-03-01

    Amyloid precursor protein (APP) fragment containing amino acids 667-676, (APP₆₆₇₋₆₇₆), is a substrate for β-secretase which is responsible for generating amyloid β peptides. Conformational analysis of APP₆₆₇₋₆₇₆ peptide [Ac-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂] and the effect of substitution of Asp₆₇₂ with D-Asp and iso-L-Asp, studied for the first time, demonstrate that the peptide backbone of APP₆₆₇₋₆₇₆ is flexible and adopts different conformations in different solvent environments (water, trifluoroethanol and dimethylsulfoxide). A major conformational difference was observed in trifluoroethanol solvent when Asp₆₇₂ is substituted with D-Asp and iso-Asp. These conformational changes involved in APP₆₆₇₋₆₇₆ may assist in understanding the interactions between β-secretase and APP₆₆₇₋₆₇₆, with relevance to Alzheimer's disease.

  14. Novel germline p16(INK4) allele (Asp145Cys) in a family with multiple pancreatic carcinomas. Mutations in brief no. 148. Online.

    PubMed

    Moskaluk, C A; Hruban, H; Lietman, A; Smyrk, T; Fusaro, L; Fusaro, R; Lynch, J; Yeo, C J; Jackson, C E; Lynch, H T; Kern, S E

    1998-01-01

    As part of a search for causative genes of familial pancreatic carcinoma, the p16 genes were sequenced in members of 21 families with a phenotype of familial pancreatic carcinoma (2 or more first degree relatives affected). One family was found in which members carried a novel p16 allele with a G to T transversion at position 451, creating a missense amino acid change at codon 145 (Asp to Cys) and possibly disrupting the donor splice site of the exon 2/3 boundary. This coding change is not a known polymorphism, and occurs at a codon position in which another missese/splicing change has been shown to be linked to familial melanoma/pancreas cancer.

  15. Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

    PubMed

    Wang, Huanchen; Nair, Vasudha S; Holland, Ashley A; Capolicchio, Samanta; Jessen, Henning J; Johnson, Michael K; Shears, Stephen B

    2015-10-27

    Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

  16. Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

    PubMed Central

    Ekici, Özlem Doğan; Paetzel, Mark; Dalbey, Ross E.

    2008-01-01

    Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called “classical” catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of “nonclassical” serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class. PMID:18824507

  17. One-step formation of oligopeptide-like molecules from Glu and Asp in hydrothermal environments.

    PubMed

    Kawamura, Kunio; Shimahashi, Masanori

    2008-05-01

    Biopolymer accumulation in the absence of enzymes is an essential step for the chemical evolution of primitive life-like systems, and successful simulation experiments of prebiotic biopolymer formation have suggested that oligopeptides could have formed near submarine hydrothermal vent environments on primitive earth. However, the yield and length of oligopeptides -- typically limited to 6-mers -- seems to be inadequate. One reason is the rapid formation of diketopiperazines (DKPs) from dipeptides. In this study, using a hydrothermal microflow reactor, we show that the one-step synthesis of oligopeptide-like molecules of length up to 20-mers proceeds from Glu and Asp. Yields of up to 0.17-0.57% were obtained in an acidic solution within 183 s at 250-310 degrees C, as evaluated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis and different types of high-performance liquid chromatography (HPLC) analyses. The present study indicates that Glu and Asp may have played important roles in the chemical evolution of oligopeptide-like molecules in hydrothermal vent environments on primitive earth. PMID:18253712

  18. Planning and Implementation of an Alkali-Surfactant-Polymer (ASP) Field Project

    SciTech Connect

    French, T.

    1999-03-05

    The Warden ASP project has progressed from the initial planning stage to construction of an injection plant. An ASP chemical system was designed based on laboratory evaluations that included interfacial tension, mobility requirements, rock-alkali interaction, fluid capabilities, and core tests. Field cores were obtained from the Permian No. 5 and No. 6 sands on the Warden lease in Sho-Vel-Tum oil field. A separate tank battery for the pilot pattern area was installed, and a field tracer test is currently being evaluated. Tracer test results to date indicate that there is no major fracturing in the No. 5 sand. There is indication, however, of some channeling through high permeability sand. The field injection plant was designed, and construction is in progress. Several variations of injection plant design have been evaluated. Some plant design details, such as alkali storage, were found to be dependent on the availability of use equipment and project budget. The surfactant storage facility design was shown to be dependent on surfactant rheology.

  19. Comparison of taurine, GABA, Glu, and Asp as scavengers of malondialdehyde in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Deng, Yan; Wang, Wei; Yu, Pingfeng; Xi, Zhijiang; Xu, Lijian; Li, Xiaolong; He, Nongyue

    2013-04-01

    The purpose of this study is to determine if amino acid neurotransmitters such as gamma-aminobutyric acid (GABA), taurine, glutamate (Glu), and aspartate (Asp) can scavenge activated carbonyl toxicants. In vitro, direct reaction between malondialdehyde (MDA) and amino acids was researched using different analytical methods. The results indicated that scavenging activated carbonyl function of taurine and GABA is very strong and that of Glu and Asp is very weak in pathophysiological situations. The results provided perspective into the reaction mechanism of taurine and GABA as targets of activated carbonyl such as MDA in protecting nerve terminals. In vivo, we studied the effect of taurine and GABA as antioxidants by detecting MDA concentration and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. It was shown that MDA concentration was decreased significantly, and the activities of SOD and GSH-Px were increased significantly in the cerebral cortex and hippocampus of acute epileptic state rats, after the administration of taurine and GABA. The results indicated that the peripherally administered taurine and GABA can scavenge free radicals and protect the tissue against activated carbonyl in vivo and in vitro.

  20. Evaluation of architectures for an ASP MPEG-4 decoder using a system-level design methodology

    NASA Astrophysics Data System (ADS)

    Garcia, Luz; Reyes, Victor; Barreto, Dacil; Marrero, Gustavo; Bautista, Tomas; Nunez, Antonio

    2005-06-01

    Trends in multimedia consumer electronics, digital video and audio, aim to reach users through low-cost mobile devices connected to data broadcasting networks with limited bandwidth. An emergent broadcasting network is the digital audio broadcasting network (DAB) which provides CD quality audio transmission together with robustness and efficiency techniques to allow good quality reception in motion conditions. This paper focuses on the system-level evaluation of different architectural options to allow low bandwidth digital video reception over DAB, based on video compression techniques. Profiling and design space exploration techniques are applied over the ASP MPEG-4 decoder in order to find out the best HW/SW partition given the application and platform constraints. An innovative SystemC-based system-level design tool, called CASSE, is being used for modelling, exploration and evaluation of different ASP MPEG-4 decoder HW/SW partitions. System-level trade offs and quantitative data derived from this analysis are also presented in this work.

  1. The Fourth Joint Meeting of the AAS and ASP, Victoria, June 1952

    NASA Astrophysics Data System (ADS)

    Garstang, R. H.

    1999-05-01

    This meeting was my most memorable because (1) it was the first AAS meeting to be held in Western Canada, (2) it was my first visit to Canada, (3) it was the occasion of the dedication of the Dominion Astrophysical Observatory 72-inch telescope to the memory of J. S. Plaskett, in the presence of Mrs. Plaskett, (4) there was a special symposium on emission line stars, which included a lecture by Paul Merrill to celebrate his retirement, and C. S. Beals and J. A. Rottenberg as the other main speakers, (5) Robert McMath gave the first Aitken lecture in memory of the famous double star astronomer, (6) I was elected a member of the AAS by the Council at this meeting, (7) I was already a member of the ASP, and this was my first opportunity to present a paper at an ASP meeting, and I could choose to put my abstract in the P.A.S.P., (8) I met many astronomers there who have been friends ever since, (9) along with many attendees I visited Butchart's Gardens, and (10) there was an excellent banquet at the Empress Hotel.

  2. Project Overview: Cumulus Humilis Aerosol Processing Study (CHAPS): Proposed Summer 2007 ASP Field Campaign

    SciTech Connect

    Berkowitz, Carl M.; Berg, Larry K.; Ogren, J. A.; Hostetler, Chris A.; Ferrare, Richard

    2006-05-18

    This white paper presents the scientific motivation and preliminary logistical plans for a proposed ASP field campaign to be carried out in the summer of 2007. The primary objective of this campaign is to use the DOE Gulfstream-1 aircraft to make measurements characterizing the chemical, physical and optical properties of aerosols below, within and above large fields of fair weather cumulus and to use the NASA Langley Research Center’s High Spectral Resolution Lidar (HSRL) to make independent measurements of aerosol backscatter and extinction profiles in the vicinity of these fields. Separate from the science questions to be addressed by these observations will be information to add in the development of a parameterized cumulus scheme capable of including multiple cloud fields within a regional or global scale model. We will also be able to compare and contrast the cloud and aerosol properties within and outside the Oklahoma City plume to study aerosol processes within individual clouds. Preliminary discussions with the Cloud and Land Surface Interaction Campaign (CLASIC) science team have identified overlap between the science questions posed for the CLASIC Intensive Operation Period (IOP) and the proposed ASP campaign, suggesting collaboration would benefit both teams.

  3. Microwave Surface Impedance Measurements of SrFe2(As,P)2 Single Crystals

    NASA Astrophysics Data System (ADS)

    Takahashi, Hideyuki; Imai, Yoshinori; Maeda, Atsutaka; Kitagawa, Kentaro; Matsubayashi, Kazuyuki; Takigawa, Masashi; Uwatoko, Yoshiya

    2012-02-01

    Various pairing symmetries have been proposed concerning Fe-based superconductors both theoretically and experimentally. It was reported that LaFePO[1] and BaFe2(As,P)2[2] have line nodes in their superconducting gap. It is in sharp contrast to other Fe-based compounds such as LiFeAs[3] and Fe(Se,Te)[4]. To confirm whether line nodes in gap function is a common feature among P doped systems, we measured the microwave surface impedances of SrFe2(As,P)2 single crystals (Tc˜30K). Single crystals were grown by self-flux method. The surface impedances were measured using a cavity perturbation technique. The imaginary part of surface impedance, which is proportional to London penetration depth in the superconducting state, shows a power law, λ(T)-λ(0)T^n. The power law indicates low-energy quasiparticle excitation, and an exponent slightly smaller than 2 does not exclude the possibility of the existence of line nodes. [ 1 ] J. D. Fletcher et al., Phys. Rev. Lett. 102 (2009) 147001.[ 2 ] K. Hashimoto et al., Phys. Rev. B 81 (2010) 220501(R).[ 3 ] Y. Imai et al., J. Phys. Soc. Jpn. 80 (2011) 013704.[ 4 ] H. Takahashi et al., Phys. Rev. B 84. (2011) 132503.

  4. Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: A theoretical analysis of structural elements

    SciTech Connect

    Bondar, A.N.; Suhai, Sandor; Fischer, S.; Smith, Jeremy C; Elstner, Marcus

    2006-10-01

    The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (1) retinal twisting; (2) hydrogen-bonding distances in the active site; and (3) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.

  5. Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: A theoretical analysis of structural elements

    SciTech Connect

    Bondar, A.N.; Suhai, Sandor; Fischer, S.; Smith, Jeremy C; Elstner, Marcus

    2007-03-01

    The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.

  6. Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: A theoretical analysis of structural elements.

    SciTech Connect

    Bondar, A.N.; Suhai, Sandor; Fischer, S.; Smith, Jeremy C; Elstner, Marcus

    2007-03-01

    The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (1) retinal twisting; (2) hydrogen-bonding distances in the active site; and (3) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.

  7. Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response.

    PubMed

    González-Hernández, Ana; Van Coppernolle, Stefanie; Borloo, Jimmy; Van Meulder, Frederik; Paerewijck, Oonagh; Peelaers, Iris; Leclercq, Georges; Claerebout, Edwin; Geldhof, Peter

    2016-01-01

    The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity. PMID:27403891

  8. Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response

    PubMed Central

    González-Hernández, Ana; Van Coppernolle, Stefanie; Borloo, Jimmy; Van Meulder, Frederik; Paerewijck, Oonagh; Peelaers, Iris; Leclercq, Georges; Claerebout, Edwin; Geldhof, Peter

    2016-01-01

    The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity. PMID:27403891

  9. New method to visualize neurons with DAT in slices of rat VTA using fluorescent substrate for DAT, ASP+

    PubMed

    Inyushin, Mikhail U; Arencibia-Albite, Francisco; de la Cruz, Angel; Vázquez-Torres, Rafael; Colon, Katiria; Sanabria, Priscila; Jiménez-Rivera, Carlos A

    2013-04-01

    The ventral tegmental area (VTA), and in particular dopamine (DA) neurons in this region of midbrain, has been shown to play an important role in motivation (goal-directed behavior), reward, and drug addiction. Most evidence that implicates VTA DA neurons in these functions are based on widely accepted but indirect electrophysiological characterization, including the hyperpolarization activated non-specific cation current (Ih), spike frequency, and inhibition by D2 receptor agonists. In this study, we used a known neuronal dopamine transporter (DAT) fluorescent substrate [4-(4- (dimethylamino) styryl)-N-methylpyridinium iodide] (ASP+) to visualize DAT-containing cell bodies of DA neurons in VTA region in rat brain slices. Uptake of 100 nM of ASP+ in brain slices of rat VTA region marked 38% of visible neurons, while other neurons from this region and 100% neurons from hippocampus slices were not fluorescent. Using patch-clamp techniques, we have found that pronounced Ih current was present in all fluorescent neurons from VTA area, also spike frequency was similar to the widely accepted values for DA neurons. Furthermore, additional study has shown that there are 84% coincidence of ASP+ fluorescence in neuronal cell bodies and Falck-Hillarp labeling of DA cells. Electrophysiological recordings during ASP+ application have confirmed that low concentrations (100 nM) of ASP+ have no visible effect on neuronal activity during 1-2 hours after staining. Thus, uptake of fluorescent monoamine analog ASP+ by DAT can be an additional criterion for identification of DAT-containing neurons in slices.

  10. Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation.

    PubMed

    Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi

    2015-01-01

    Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology.

  11. The Asp245-->Asn mutant of Coprinus cinereus peroxidase. Characterization by 1H-NMR spectroscopy and comparison with the wild-type enzyme.

    PubMed

    Veitch, N C; Gao, Y; Welinder, K G

    1996-11-12

    The resting, fluoride-ligated and cyanide-ligated states of the Asp245-->Asn mutant of Coprinus cinereus peroxidase (D245N CIP) have been characterized using 1H-NMR spectroscopy in conjunction with parallel studies of the wild-type enzyme. Analysis of the spectra of resting state D245N CIP over the pH range 5-10 has uncovered the existence of three high-spin species in dynamic equilibrium with each other. The predominant species at neutral pH is six-coordinate high-spin (6-c HS), with a distal water molecule as the sixth ligand. This species is in slow exchange on the NMR time scale with a second six-coordinate high-spin species (6-c HS*) and a five-coordinate high-spin species (5-c HS**), toward acidic and alkaline pH values, respectively. The 6-c HS* species appears to be unique and is proposed to differ from the 6-c HS species by protonation of the proximal His residue, whereas the 5-c HS** species lacks the proximal His ligand and is coordinated by a hydroxyl group. In sharp contrast, wild-type CIP is a five-coordinate high-spin (5-c HS) species over the same pH range. The D245N CIP mutant also exhibits a greater affinity for fluoride than wild-type CIP. The 1H-NMR spectrum of cyanide-ligated D245N CIP, assigned using two-dimensional methods, differs significantly from that of the wild-type enzyme. Perturbations to heme and heme-linked proton resonances are rationalised in terms of the loss or significant weakening of the hydrogen bond between His183 N delta 1H and the side-chain of residue 245 when Asp is replaced by Asn. This subtle interaction directly affects the heme pocket structure of CIP both proximal and distal to the heme plane.

  12. Association of endothelial nitric oxide synthase (eNOS) gene polymorphism (Glu 298 Asp) with coronary artery disease in subjects from Multan, Pakistan.

    PubMed

    Taqddus, Asia; Saad, Abu-Bakar Ali; Pasha, Burhan; Latif, Muhammad; Safdar, Sohail; Shaikh, Rehan Sadiq; Ali, Muhammad; Iqbal, Furhan

    2014-03-01

    Acute Coronary Syndrome (ACS) is the most common disease and cause of mortality in both genders across the world and certain risk factors i.e. age, gender, smoking, diabetes, hypertension, drugs usage, weight etc are known to be associated with the disease. The aim of this study was to find if there is any correlation exists between ACS and hereditary genetic defect in endothelial nitric oxide synthase (ecNOS) gene as eNOS generates Nitric oxide in blood vessels and regulates the vascular tone hence directly affecting the cardiovascular function. Single nucleotide polymorphism (SNP) (Glu 298 Asp) in ecNOS was determined in 280 subjects, from Southern Punjab (in Pakistan) population, including (160 ACS patients and 120 healthy controls) by PCR-RFLP method and genotype was correlated with various risk factors as well as with serum cholesterol and triglyceride levels. Our results indicated that the genotype Glu 298 Asp was not associated with ACS but when various studied parameters were compared among patients suffering from various forms of ACS and their healthy controls, it was observed that age (45-55 years) (P = 0.05), gender (male) (P < 0.001), education (P<0.001), family history (P=0.03), hypertension (P<0.001), diabetes (P<0.01) and smoking habit (P = 0.03) were the significantly different parameters among them and may be associated with the incidence of cardiovascular disease. Cholesterol (161.5±79 mg/dL) level was found to be higher in patients (P = 0.04) than controls while triglyceride remained unaffected (P = 0.87) in both groups. PMID:24577926

  13. Conceptual design of a noncontacting power transfer device for the ASPS Vernier system

    NASA Technical Reports Server (NTRS)

    Kroeger, J.; Drilling, J.; Gunderman, T.

    1984-01-01

    The conceptual of electrical power transfer across a magnetically controlled gap as discussed for several years. The design represents the culmination of the first serious attempt to design a very low force, noncontracting power transfer mechanism. The electromagnetic device advanced herein is an ironless, translatable secondary transformer in which one of the two coils is fixed to the entire magnetic core. The second coil is free to move within the core over the full range of motions required. The specific application considered for this design was the Vernier subsystem of the Annular Suspension and Pointing System (ASPS). The development of and rationale for the electromagnetics design is presented. Similar documentation is provided for the Electronics Design. The Appendices detail the results of small scale model tests, disturbance force calculations, the baseline transformer fabrication drawings, the AVS Converter Parts List, and model schematic diagrams.

  14. A novel member of the integrin receptor family mediates Arg-Gly-Asp- stimulated neutrophil phagocytosis

    PubMed Central

    1989-01-01

    Human neutrophils (PMN) express a heterodimeric receptor that has ligand binding specificity for the Arg-Gly-Asp (RGD) sequence within many adhesive proteins. A monoclonal antibody, B6H12, which binds to this receptor, inhibits both RGD-mediated ligand binding and stimulation of IgG-mediated phagocytosis by fibronectin, fibrinogen, vitronectin, von Willebrand's factor, and collagen type IV. By several criteria this receptor is neither a known very late antigen, a known cytoadhesin (gp IIb/IIIa-vitronectin receptor), nor a member of the LFA- 1, Mac-1, p150,95 group of integrin receptors. Ligand binding via this receptor is rapidly inactivated by products of the myeloperoxidase- hydrogen peroxide-halide system of PMN. We conclude that this receptor, for which we propose the name leukocyte response integrin, is a signal- transducing molecule on PMN which may have a significant early role in modulation of PMN function at inflammatory sites. PMID:2785522

  15. Genetics of rare mesenchymal tumors: implications for targeted treatment in DFSP, ASPS, CCS, GCTB and PEComa.

    PubMed

    Rutkowski, Piotr; Przybył, Joanna; Świtaj, Tomasz

    2014-08-01

    Soft tissue and bone sarcomas comprise a heterogeneous group of mesenchymal tumors that include roughly 130 distinct diagnostic entities. Many of them are exceptionally rare, with only few cases diagnosed worldwide each year. Development of novel targeted treatment in this group of tumors is of special importance since many sarcoma subtypes are resistant to conventional chemotherapy and the effective therapeutic options are limited. In this review we aim to discuss the molecular implications for targeted therapy in selected rare soft tissue and bone sarcoma subtypes, including dermatofibrosarcoma protuberans (DFSP), alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), giant cell tumor of bone (GCTB) and perivascular epithelioid cell neoplasms (PEComas). This article is part of a Directed Issue entitled: Rare cancers.

  16. Radical formation of amino acid precursors in interstellar regions? Ser, Cys and Asp.

    PubMed

    Knowles, Daniel J; Wang, Tianfang; Bowie, John H

    2010-11-01

    It is proposed that the glycine precursor NH(2)CH(2)CN may be synthesised in interstellar dust clouds by the radical combination reactions NH(2)˙ + ˙CH(2)CN → NH(2)CH(2)CN (ΔG = -302 kJ mol(-1)) and/or NH(2)CH(2)˙ + ˙CN → NH(2)CH(2)CN (ΔG = -414 kJ mol(-1)). All calculations at the CCSD(T)/aug-cc-pVDZ//B3LYP/6-31+G(d) level of theory. This paper extends that concept to radical/radical coupling reactions to form Ser, Cys and Asp precursor nitriles. The hydrogen abstraction process NH(2)CH(2)CN + HO˙→ NH(2)˙CHCN + H(2)O (ΔG = -130 kJ mol(-1)) is suggested to precede the radical coupling reactions NH(2)˙CHCN + R˙→ NH(2)CHRCN (R˙ = ˙CH(2)OH, ˙CH(2)SH and ˙CH(2)CN) to form nitrile precursors of the amino acids Ser, Cys and Asp. These three reactions are all favourable (ΔG = -240, -227 and -223 kJ mol(-1)). The radical species ˙CH(2)NH(2), ˙CH(2)OH, ˙CH(2)SH and ˙CH(2)CN are shown to be stable for the microsecond timeframe by a combination of theoretical calculations and the experimental mass spectrometric neutralization/reionization procedure.

  17. Conformational consequences of ionization of Lys, Asp, and Glu buried at position 66 in staphylococcal nuclease.

    PubMed

    Karp, Daniel A; Stahley, Mary R; García-Moreno, Bertrand

    2010-05-18

    The pK(a) values measured previously for the internal Lys-66, Asp-66, and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pK(a) units relative to normal pK(a) values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of the ionizable moieties in crystal structures. To examine the origins of these high apparent dielectric constants, we showed that the pK(a) values of these internal residues are sensitive to the global stability of the protein; the shifts tend to be smaller in less stable forms of nuclease. This implies that the apparent dielectric constants reported by these internal ionizable groups are high because they reflect conformational reorganization coupled to their ionization. To detect this directly, acid-base titrations monitored with Trp fluorescence and near-UV and far-UV CD spectroscopy were performed on variants with Lys-66, Glu-66, or Asp-66 in background proteins with different stabilities. Conformational reorganization coupled to the ionization of the internal groups was spectroscopically detectable, especially in the less stable background proteins. The data show that to improve the accuracy of structure-based pK(a) calculations of internal groups the calculations will have to treat explicitly all structural reorganization coupled to ionization. The data also suggest a novel approach to mapping the folding free energy landscape of proteins by using internal ionizable groups to stabilize partially unfolded states.

  18. IL6R Variation Asp358Ala Is a Potential Modifier of Lung Function in Asthma

    PubMed Central

    Hawkins, Gregory A; Robinson, Mac B; Hastie, Annette T; Li, Xingnan; Li, Huashi; Moore, Wendy C; Howard, Timothy D; Busse, William W.; Erzurum, Serpil C.; Wenzel, Sally E.; Peters, Stephen P; Meyers, Deborah A; Bleecker, Eugene R

    2012-01-01

    Background The IL6R SNP rs4129267 has recently been identified as an asthma susceptibility locus in subjects of European ancestry but has not been characterized with respect to asthma severity. The SNP rs4129267 is in linkage disequilibrium (r2=1) with the IL6R coding SNP rs2228145 (Asp358Ala). This IL6R coding change increases IL6 receptor shedding and promotes IL6 transsignaling. Objectives To evaluate the IL6R SNP rs2228145 with respect to asthma severity phenotypes. Methods The IL6R SNP rs2228145 was evaluated in subjects of European ancestry with asthma from the Severe Asthma Research Program (SARP). Lung function associations were replicated in the Collaborative Study on the Genetics of Asthma (CSGA) cohort. Serum soluble IL6 receptor (sIL6R) levels were measured in subjects from SARP. Immunohistochemistry was used to qualitatively evaluate IL6R protein expression in BAL cells and endobronchial biopsies. Results The minor C allele of IL6R SNP rs2228145 was associated with lower ppFEV1 in the SARP cohort (p=0.005), the CSGA cohort (0.008), and in combined cohort analysis (p=0.003). Additional associations with ppFVC, FEV1/FVC, and PC20 were observed. The rs2228145 C allele (Ala358) was more frequent in severe asthma phenotypic clusters. Elevated serum sIL6R was associated with lower ppFEV1 (p=0.02) and lower ppFVC (p=0.008) (N=146). IL6R protein expression was observed in BAL macrophages, airway epithelium, vascular endothelium, and airway smooth muscle. Conclusions The IL6R coding SNP rs2228145 (Asp358Ala) is a potential modifier of lung function in asthma and may identify subjects at risk for more severe asthma. IL6 transsignaling may have a pathogenic role in the lung. PMID:22554704

  19. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  20. An Asp-CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells.

    PubMed

    Schoborg, Todd; Zajac, Allison L; Fagerstrom, Carey J; Guillen, Rodrigo X; Rusan, Nasser M

    2015-12-01

    The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure-function analysis support this hypothesis. Disruption of the Asp-CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp's spindle function; however, it is completely dispensable for Asp's role in microcephaly suppression. PMID:26620907

  1. Vacuolar protein sorting 35 Asp620Asn mutation is rare in the ethnic Chinese population with Parkinson's disease.

    PubMed

    Zhang, Yu; Chen, Shuai; Xiao, Qin; Cao, Li; Liu, Jun; Rong, Tian-Yi; Ma, Jian-Fang; Wang, Gang; Wang, Ying; Chen, Sheng-Di

    2012-06-01

    Vacuolar protein sorting 35 (VPS35) Asp620Asn mutation has been identified in late-onset familial Parkinson's disease (PD) patients of Swiss and Austrian descent as well as sporadic PD patients in the United States. In order to determine the contribution of VPS35 mutations in mainland Chinese PD patients and to better understand the association between VPS35 and PD, we sequenced all 17 exons of VPS35 in 32 probands of presumed autosomal-dominant, late-onset familial PD and 35 normal controls. Meanwhile, we analyzed VPS35 Asp620Asn mutation in 512 PD patients. A total of 371 subjects without neurological disorders from the same region in China were set as a control group. We did not find any VPS35 coding region mutation in 32 familial PD patients. VPS35 Asp620Asn mutation was either not found in 480 PD patients. Our results suggested that VPS35 Asp620Asn may be not associated with PD in Chinese population.

  2. Bovine rhodopsin: amino acid substitutions Asp-83----Asn and Glu-134----Gln prevent activation of cyclic GMP phosphodiesterase.

    PubMed

    Gurevich, V V; Zozulya, S A; Zerf, E P; Pokrovskaya, I D; Obukhova, T A; Garnovskaya, M N; Dumler, I L; Rychkova, M P

    1990-01-01

    Two bovine rhodopsin mutants with substitutions of negatively charged residues within transmembrane domains II and III by uncharged ones (Asp-83----Asn and Glu-134----Gln) were constructed. Both mutants stimulated transducin GTPase with slightly lowered efficiency, but were completely unable to activate cyclic GMP phosphodiesterase. PMID:1966786

  3. Clinical experiences utilizing wireless remote control and an ASP model backup archive for a disaster recovery event

    NASA Astrophysics Data System (ADS)

    Liu, Brent J.; Documet, Luis; Documet, Jorge; Huang, H. K.; Muldoon, Jean

    2004-04-01

    An Application Service Provider (ASP) archive model for disaster recovery for Saint John"s Health Center (SJHC) clinical PACS data has been implemented using a Fault-Tolerant Archive Server at the Image Processing and Informatics Laboratory, Marina del Rey, CA (IPIL) since mid-2002. The purpose of this paper is to provide clinical experiences with the implementation of an ASP model backup archive in conjunction with handheld wireless technologies for a particular disaster recovery scenario, an earthquake, in which the local PACS archive and the hospital are destroyed and the patients are moved from one hospital to another. The three sites involved are: (1) SJHC, the simulated disaster site; (2) IPIL, the ASP backup archive site; and (3) University of California, Los Angeles Medical Center (UCLA), the relocated patient site. An ASP backup archive has been established at IPIL to receive clinical PACS images daily using a T1 line from SJHC for backup and disaster recovery storage. Procedures were established to test the network connectivity and data integrity on a regular basis. In a given disaster scenario where the local PACS archive has been destroyed and the patients need to be moved to a second hospital, a wireless handheld device such as a Personal Digital Assistant (PDA) can be utilized to route images to the second hospital site with a PACS and reviewed by radiologists. To simulate this disaster scenario, a wireless network was implemented within the clinical environment in all three sites: SJHC, IPIL, and UCLA. Upon executing the disaster scenario, the SJHC PACS archive server simulates a downtime disaster event. Using the PDA, the radiologist at UCLA can query the ASP backup archive server at IPIL for PACS images and route them directly to UCLA. Implementation experiences integrating this solution within the three clinical environments as well as the wireless performance are discussed. A clinical downtime disaster scenario was implemented and successfully

  4. Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

    PubMed

    He, Yuxian; Barker, Sophie J; MacDonald, Angus J; Yu, Yu; Cao, Long; Li, Jingjing; Parhar, Ranjit; Heck, Susanne; Hartmann, Susanne; Golenbock, Douglas T; Jiang, Shibo; Libri, Nathan A; Semper, Amanda E; Rosenberg, William M; Lustigman, Sara

    2009-04-01

    We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant. PMID:19299698

  5. Antigen Sparing and Enhanced Protection Using A Novel rOv-ASP-1 Adjuvant in Aqueous Formulation with Influenza Vaccines

    PubMed Central

    Jiang, Jiu; Fisher, Erin M.; Hensley, Scott E.; Lustigman, Sara; Murasko, Donna M.; Shen, Hao

    2014-01-01

    Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: 1) accelerates and enhances the specific antibody response induced by influenza vaccine; 2) allows for antigen sparing; and 3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain. PMID:24681229

  6. Nuclear-targeting TAT-PEG-Asp8-doxorubicin polymeric nanoassembly to overcome drug-resistant colon cancer

    PubMed Central

    Pan, Zhen-zhen; Wang, Hui-yuan; Zhang, Meng; Lin, Ting-ting; Zhang, Wen-yuan; Zhao, Peng-fei; Tang, Yi-si; Xiong, Yong; Zeng, Yuan-er; Huang, Yong-zhuo

    2016-01-01

    Aim: Drug efflux-associated multidrug resistance (MDR) is a main obstacle to effective cancer chemotherapy. Large molecule drugs are not the substrates of P-glycoprotein, and can circumvent drug efflux and be retained inside cells. In this article we report a polymer-drug conjugate nanoparticulate system that can overcome MDR based on size-related exclusion effect. Methods: Doxorubicin was coupled with the triblock polymeric material cell-penetrating TAT-PEG-poly(aspartic acid). The amphiphilic macromolecules (termed TAT-PEG-Asp8-Dox) could self-assemble into nanoparticles (NPs) in water. The antitumor activity was evaluated in drug-resistant human colon cancer HCT8/ADR cells in vitro and in nude mice bearing HCT8/ADR tumor. Results: The self-assembling TAT-PEG-Asp8-Dox NPs were approximately 150 nm with a narrow particle size distribution, which not only increased the cellular uptake efficiency, but also bypassed P-glycoprotein-mediated drug efflux and improved the intracellular drug retention, thus yielding an enhanced efficacy for killing drug-resistant HCT8/ADR colon cancer cells in vitro. Importantly, the TAT-PEG-Asp8-Dox NPs enhanced the intranuclear disposition of drugs for grater inhibition of DNA/RNA biosynthesis. In nude mice bearing xenografted HCT8/ADR colon cancers, intravenous or peritumoral injection of TAT-PEG-Asp8-Dox NPs for 22 d effectively inhibited tumor growth. Conclusion: TAT-PEG-Asp8-Dox NPs can increase cellular drug uptake and intranuclear drug delivery and retain effective drug accumulation inside the cells, thus exhibiting enhanced anticancer activity toward the drug-resistant human colon cancer HCT8/ADR cells. PMID:27292613

  7. Antigen sparing and enhanced protection using a novel rOv-ASP-1 adjuvant in aqueous formulation with influenza vaccines.

    PubMed

    Jiang, Jiu; Fisher, Erin M; Hensley, Scott E; Lustigman, Sara; Murasko, Donna M; Shen, Hao

    2014-05-13

    Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: (1) accelerates and enhances the specific antibody response induced by influenza vaccine; (2) allows for antigen sparing; and (3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain.

  8. Antigen sparing and enhanced protection using a novel rOv-ASP-1 adjuvant in aqueous formulation with influenza vaccines.

    PubMed

    Jiang, Jiu; Fisher, Erin M; Hensley, Scott E; Lustigman, Sara; Murasko, Donna M; Shen, Hao

    2014-05-13

    Influenza is one of the most common infectious diseases endangering the health of humans, especially young children and the elderly. Although vaccination is the most effective means of protection against influenza, frequent mutations in viral surface antigens, low protective efficacy of the influenza vaccine in the elderly, slow production process and the potential of vaccine supply shortage during a pandemic are significant limitations of current vaccines. Adjuvants have been used to enhance the efficacy of a variety of vaccines; however, no adjuvant is included in current influenza vaccines approved in the United States. In this study, we found that a novel adjuvant, rOv-ASP-1, co-administrated with inactivated influenza vaccine using an aqueous formulation, substantially improved the influenza-specific antibody response and protection against lethal infection in a mouse model. rOv-ASP-1 enhanced the magnitude of the specific antibody response after immunization with low doses of influenza vaccine, allowing antigen-sparring by 10-fold. The rOv-ASP-1 formulated vaccine induced a more rapid response and a stronger Th1-associated antibody response compared to vaccine alone and to the vaccine formulated with the adjuvant alum. Importantly, rOv-ASP-1 significantly enhanced cross-reactive antibody responses and protection against challenge with an antigenically distinct strain. These results demonstrate that rOv-ASP-1 is an effective adjuvant that: (1) accelerates and enhances the specific antibody response induced by influenza vaccine; (2) allows for antigen sparing; and (3) augments a Th1-biased and cross-reactive antibody response that confers protection against an antigenically distinct strain. PMID:24681229

  9. Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo.

    PubMed

    Lin, Zhen-Yu; Duan, Zhi-Xia; Guo, Xiao-Dong; Li, Jing-Feng; Lu, Hong-Wei; Zheng, Qi-Xin; Quan, Da-Ping; Yang, Shu-Hua

    2010-06-01

    BMP-2 is one of the most important growth factors of bone regeneration. Polylactide-co-glycolic acid (PLGA), which is used as a biodegradable scaffold for delivering therapeutic agents, has been intensively investigated. In previous studies, we synthesized a novel BMP-2-related peptide (designated P24) and found that it could enhance the osteoblastic differentiation of bone marrow stromal cells (BMSCs). The objective of this study was to construct a biomimetic composite by incorporating P24 into a modified PLGA-(PEG-ASP)n copolymer to promote bone formation. In vitro, our results demonstrated that PLGA-(PEG-ASP)n scaffolds were shown to be an efficient system for sustained release of P24. Significantly more BMSCs attached to the P24/PLGA-(PEG-ASP)n and PLGA-(PEG-ASP)n membranes than to PLGA, and the cells in the two groups subsequently proliferated more vigorously than those in the PLGA group. The expression of osteogenic markers in P24/PLGA-(PEG-ASP)n group was stronger than that in the PLGA-(PEG-ASP)n and PLGA groups. Radiographic and histological examination, Western blotting and RT-PCR showed that P24/PLGA-(PEG-ASP)n scaffold could induce more effective ectopic bone formation in vivo, as compared with PLGA-(PEG-ASP)n or gelatin sponge alone. It is concluded that the PLGA-(PEG-ASP)n copolymer is a good P24 carrier and can serve as a good scaffold for controlled release of P24. This novel P24/PLGA-(PEG-ASP)n composite promises to be an excellent biomaterial for inducing bone regeneration.

  10. Failure to find linkage between a functional polymorphism in the dopamine D4 receptor gene and schizophrenia

    SciTech Connect

    Shaikh, S.; Gill, M.; Collier, D.A.

    1994-03-15

    We report the results of a linkage study in 24 families multiply affected with schizophrenia using a polymorphic DNA sequence encoding the third cytoplasmic loop of the dopamine D4 receptor. Two-point LOD score analyses with a range of single gene models ranging from near dominant to near recessive revealed no evidence for linkage. In addition, we examined the data by non-parametric sib-pair analysis and found no excess sharing of alleles between affected sib-pairs. We therefore conclude that mutations within the dopamine D4 receptor gene do not have a major aetiological role in schizophrenia in our collection of pedigrees. 20 refs., 2 tabs.

  11. The ALK inhibitor ASP3026 eradicates NPM-ALK⁺ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model.

    PubMed

    George, Suraj Konnath; Vishwamitra, Deeksha; Manshouri, Roxsan; Shi, Ping; Amin, Hesham M

    2014-07-30

    NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.

  12. The dipeptide Pro-Asp promotes IGF-1 secretion and expression in hepatocytes by enhancing JAK2/STAT5 signaling pathway.

    PubMed

    Wang, Songbo; Wang, Guoqing; Zhang, Mengyuan; Zhuang, Lu; Wan, Xiaojuan; Xu, Jingren; Wang, Lina; Zhu, Xiaotong; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Shu, Gang; Jiang, Qingyan

    2016-11-15

    It has been implicated that IGF-1 secretion can be regulated by dietary protein. However, whether the dipeptides, one of digested products of dietary protein, have influence on IGF-1 secretion remain largely unknown. Our study aimed to investigate the effects of the dipeptide Pro-Asp on IGF-1 secretion and expression in hepatocytes and to explore the possible underlying mechanisms. Our findings demonstrated that Pro-Asp promoted the secretion and gene expression of IGF-1 in HepG2 cells and primary porcine hepatocytes. Meanwhile, Pro-Asp activated the ERK and Akt signaling pathways, downstream of IGF-1. In addition, Pro-Asp enhanced GH-mediated JAK2/STAT5 signaling pathway, while inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Asp on IGF-1 secretion and expression. Moreover, acute injection of Pro-Asp stimulated IGF-1 expression and activated JAK2/STAT5 signaling pathway in mice liver. Together, these results suggested that the dipeptide Pro-Asp promoted IGF-1 secretion and expression in hepatocytes by enhancing GH-mediated JAK2/STAT5 signaling pathway.

  13. Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

    PubMed Central

    Asojo, Oluwatoyin A.; Loukas, Alex; Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M.; Hotez, Peter J.

    2005-01-01

    Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P21 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit. PMID:16511050

  14. Synergistic activity of amenamevir (ASP2151) with nucleoside analogs against herpes simplex virus types 1 and 2 and varicella-zoster virus.

    PubMed

    Chono, Koji; Katsumata, Kiyomitsu; Suzuki, Hiroshi; Shiraki, Kimiyasu

    2013-02-01

    ASP2151 (amenamevir) is a helicase-primase complex inhibitor with antiviral activity against herpes simplex virus HSV-1, HSV-2, and varicella-zoster virus (VZV). To assess combination therapy of ASP2151 with existing antiherpes agents against HSV-1, HSV-2, and VZV, we conducted in vitro and in vivo studies of two-drug combinations. The combination activity effect of ASP2151 with nucleoside analogs acyclovir (ACV), penciclovir (PCV), or vidarabine (VDB) was tested via plaque-reduction assay and MTS assay, and the data were analyzed using isobolograms and response surface modeling. In vivo combination therapy of ASP2151 with valaciclovir (VACV) was studied in an HSV-1-infected zosteriform spread mouse model. The antiviral activity of ASP2151 combined with ACV and PCV against ACV-susceptible HSV-1, HSV-2, and VZV showed a statistically significant synergistic effect (P<0.05). ASP2151 with VDB was observed to have additive effects against ACV-susceptible HSV-2 and synergistic effects against VZV. In the mouse model of zosteriform spread, the inhibition of disease progression via combination therapy was more potent than that of either drugs as monotherapy (P<0.05). These results indicate that the combination therapies of ASP2151 with ACV and PCV have synergistic antiherpes effects against HSV and VZV infections and may be feasible in case of severe disease, such as herpes encephalitis or in patients with immunosuppression.

  15. Efficacy of ASP2151, a helicase-primase inhibitor, against thymidine kinase-deficient herpes simplex virus type 2 infection in vitro and in vivo.

    PubMed

    Himaki, Takehiro; Masui, Yumi; Chono, Koji; Daikoku, Tohru; Takemoto, Masaya; Haixia, Bo; Okuda, Tomoko; Suzuki, Hiroshi; Shiraki, Kimiyasu

    2012-02-01

    ASP2151 was developed as a novel inhibitor of herpes simplex virus (HSV) and varicella-zoster virus helicase-primase. The anti-HSV activity of ASP2151 toward a clinical HSV isolate with acyclovir (ACV)-resistant/thymidine kinase (TK)-deficiency was characterized in vitro and in vivo using a plaque reduction assay and the ear pinna infection in mice. The IC(50) ranged from 0.018 to 0.024 μg/ml, indicating the susceptibility of TK-deficient HSV-2 was similar to that of wild-type HSV-2 strains. Anti-HSV activity of ASP2151 in vivo was evaluated in mice infected with wild-type HSV-2 and TK-deficient HSV-2. ASP2151 significantly reduced the copy numbers of wild-type HSV-2 and TK-deficient HSV-2 at the inoculation ear pinna, while valacyclovir significantly reduced the copy number of wild type HSV-2 but not that of TK-deficient HSV-2 in the inoculated ear pinna. Thus, ASP 2151 showed therapeutic efficacy in mice infected with both wild-type and TK-deficient HSV-2. In conclusion, ASP2151 is a promising novel herpes helicase-primase inhibitor that indicates the feasibility of ASP2151 for clinical application for the treatment of HSV infections, including ACV-resistant/TK-deficient HSV infection.

  16. The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

    PubMed

    Muñoz, Fernando F; Mendieta, Julieta R; Pagano, Mariana R; Paggi, Roberto A; Daleo, Gustavo R; Guevara, María G

    2010-05-01

    Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

  17. Occurrence and fate of endocrine disrupting chemicals in ASP based sewage treatment plant in Hardwar.

    PubMed

    Saini, Gita; Pant, Shalini; Alam, Tanveer; Kazmi, A A

    2016-01-01

    The occurrence of emerging contaminants such as endocrine disrupting chemicals (EDCs) in our water resources is of prime concern. With this context, fate and seasonal variation of six EDCs (testosterone, T; progesterone, P; diethyl phthalate, DEP; dibutyl phthalate, DBP; propyl-paraben, PP and butyl-paraben, BP) were assessed throughout the year, i.e. in rainy, winter, spring and summer seasons in the raw, treated wastewater and activated sludge in an activated sludge process (ASP) based sewage treatment plant (STP) located in Haridwar, India. Qualitative and quantitative measurements were performed by gas chromatography-mass spectrometry (GC-MS) analysis. Results indicate that in summer, the examined STP could effectively remove 82.9% of T, 86.4% of P, 95.5% of DEP, 92.4% of DBP, 91.5% of PP, and 89.9% of BP from the wastewater. Among the EDCs considered, higher removal efficiencies were achieved for phthalates in the summer season. GC-MS analysis showed that a small fraction of EDCs was sorbed on the solid fraction of activated sludge. Scanning electron microscopy, energy dispersive X-ray spectroscopy and Fourier transformation infrared spectroscopy analysis were also performed to investigate the occurrence of EDCs in biomass samples. Results of this study also demonstrated that removal efficiency, assessed in terms of physicochemical and microbiological parameters, was maximum in summer and reached minimum in rainy season. PMID:27642823

  18. Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

    SciTech Connect

    Asojo, Oluwatoyin A.; Loukas, Alex; Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M.; Hotez, Peter J.

    2005-04-01

    In order to clarify the structural basis of the pathogenesis-related-1 domain, Na-ASP-1, the first multi-domain ASP from the human hookworm parasite N. americanus, has been crystallized. 2.2 Å resolution data have been collected from a crystal belonging to the monoclinic space group P2{sub 1}. Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P2{sub 1} with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit.

  19. A salt bridge between Arg-20 on parathyroid hormone (PTH) and Asp-137 on the PTH1 receptor is essential for full affinity.

    PubMed

    Weaver, Richard E; Wigglesworth, Mark J; Donnelly, Dan

    2014-11-01

    Parathyroid hormone (PTH) acts via the receptor PTH1 and plays an important role in calcium homeostasis. PTH's interaction with the N-terminal domain of PTH1 is mediated in part by Arg-20 on the peptide which forms a number of interactions with the receptor: a charge-charge interaction with Asp-137; hydrogen bonds with the backbone of Asp-29 and Met-32; and hydrophobic interactions with Met-32 and Gln-37. The aim of this work was to establish the importance of the charge-charge interaction through the combined use of modified peptide ligands, site-directed mutations of the receptor, and pharmacological assays. The substitution of Arg-20 with norleucine resulted in a 50-fold reduction in potency at PTH1 and Asp-137-Glu while, in contrast, both Asp-137-Asn and Asp-137-Ala receptors were largely insensitive to this ligand modification. The effect of this removal of the positive charge as position 20 could be partially rescued at PTH1 and Asp-137-Glu, but not Asp-137-Asn and Asp-137-Ala, through a substitution of peptide position 20 with ornithine. The latter two receptors, which have no negative charge at position 137, displayed potency for PTH that was reduced by 40- and 117-fold, respectively. These data demonstrate that a negative charge at residue-137 is important for interacting with ligands containing a positive charge at residue-20, and that the Arg-20 interaction with Asp-137, observed in the crystal structure of the isolated N-terminal domain of PTH1, is likely to be present in the full length receptor where it provides an important affinity- and potency-generating interaction through a salt bridge.

  20. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2014-11-13

    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  1. Pyrimidinopyrimidine inhibitors of ketohexokinase: exploring the ring C2 group that interacts with Asp-27B in the ligand binding pocket.

    PubMed

    Maryanoff, Bruce E; O'Neill, John C; McComsey, David F; Yabut, Stephen C; Luci, Diane K; Gibbs, Alan C; Connelly, Margery A

    2012-08-15

    Inhibitors of ketohexokinase (KHK) have potential for the treatment of diabetes and obesity. We have continued studies on a pyrimidinopyrimidine series of potent KHK inhibitors by exploring the 2-position substituent (R(3)) that interacts with Asp-27B in the ATP-binding region of KHK (viz. 1, 2; Table 1). We found that increased spacing between the terminal ammonium group and the heterocyclic scaffold (viz. 16-20), such that interaction with Asp-27B is not possible, still results in potent KHK inhibition (IC(50)=15-50 nM). We propose a new interaction with Asp-194, which serves to expand the pyrimidinopyrimidine pharmacophore.

  2. Evaluation of the Sho-Vel-Tum Alkali-Surfactant-Polymer (ASP) Oil Recovery Project - Stephens County, OK

    SciTech Connect

    French, Troy

    1999-08-16

    Le Norman Energy Company conducted research on field application of alkaline-surfactant-polymer (ASP) flooding as a part of the U.S. Department of Energy's plan to maximize the production of our domestic oil resources. In addition to having substantial technical merit, the process uses chemicals that are environmentally acceptable. Le Norman's field project is located in the Sho-Vel-Tum (OK) oil field, which was a major producer of crude oil in past years, but has since been extensively waterflooded. This reservoir in this portion of the field is typical of many shallow reservoirs in the Oklahoma-Kansas area and is a good demonstration site for that area. The pay zones are located approximately 700 ft. deep, and this project is the shallowest field test for ASP flooding.

  3. Synthesis of a precursor tripeptide Z-Asp-Val-Tyr-OH of thymopentin by chemo-enzymatic method.

    PubMed

    Zheng, Kun; Zhan, Ruiyan; Hong, Yang; Li, Jing; Shi, Wei; Li, Shijun

    2012-01-01

    The precursor tripeptide of thymopentin was synthesized by a combination of chemical and enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial alkaline protease alcalase was used in water-organic cosolvent systems. The synthesis reaction conditions were optimized by examining the effects of several factors including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum condition is of pH 10.0, 35°C, acetonitrile/Na₂CO₃-NaHCO₃ buffer system (85:15, v/v), and reaction time of 2.5 hr, which achieves tripeptide yield of more than 70%. PMID:23030464

  4. Synthesis of a precursor tripeptide Z-Asp-Val-Tyr-OH of thymopentin by chemo-enzymatic method.

    PubMed

    Zheng, Kun; Zhan, Ruiyan; Hong, Yang; Li, Jing; Shi, Wei; Li, Shijun

    2012-01-01

    The precursor tripeptide of thymopentin was synthesized by a combination of chemical and enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial alkaline protease alcalase was used in water-organic cosolvent systems. The synthesis reaction conditions were optimized by examining the effects of several factors including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum condition is of pH 10.0, 35°C, acetonitrile/Na₂CO₃-NaHCO₃ buffer system (85:15, v/v), and reaction time of 2.5 hr, which achieves tripeptide yield of more than 70%.

  5. Characterisation of Alternaria alternata manganese-dependent superoxide dismutase, a cross-reactive allergen homologue to Asp f 6.

    PubMed

    Gabriel, Marta F; Postigo, Idoia; Gutiérrez-Rodríguez, Antonio; Suñén, Ester; Guisantes, Jorge; Tomaz, Cândida T; Martínez, Jorge

    2015-07-01

    It is well known that Alternaria alternata presents a significant level of allergenic cross-reactivity with several other phylogenetically related and non-related allergenic moulds. To improve the molecular diagnosis, the identification and characterisation of all clinically relevant allergens, including both species-specific and cross-reacting proteins, is required. In this study we report the molecular and immunological characterisation of the A. alternata manganese-dependent superoxide dismutase (Alt a MnSOD) and its cross-reactivity with Asp f 6, a diagnostic marker allergen in allergic bronchopulmonary aspergillosis (ABPA). The cDNA coding for Alt a MnSOD sequence was isolated by RACE and PCR. Alt a MnSOD is a protein of 191 amino acids that presented significant homology and potential cross-reactive epitopes with Asp f 6. The recombinant protein was produced in Escherichia coli and the immunoreactivity was evaluated in patient sera. Immunoblotting analyses showed that seven of sixty-one A. alternata-sensitised patient sera and two ABPA patient sera reacted with the recombinant Alt a MnSOD. The native counterpart contained in both A. alternata and Aspergillus fumigatus extracts inhibited IgE binding to the recombinant molecule. The allergen was named Alt a 14 by the official Allergen nomenclature subcommittee. Thus, Alt a 14 is a relevant allergen in A. alternata sensitisation that may be used to improve diagnostic procedures. Evidence of cross-reactivity between Asp f 6 and Alt a 14-recognition by ABPA patient sera suggest the existence of an Alt a 14-mediated mechanism that, similar to Asp f 6, may be related to the pathogenesis of ABPA.

  6. Structural and Phylogenetic Analysis of a Conserved Actinobacteria-Specific Protein (ASP1; SCO1997) from Streptomyces Coelicolor

    SciTech Connect

    Gao, B.; Sugiman-Marangos, S; Junop, M; Gupta, R

    2009-01-01

    The Actinobacteria phylum represents one of the largest and most diverse groups of bacteria, encompassing many important and well-characterized organisms including Streptomyces, Bifidobacterium, Corynebacterium and Mycobacterium. Members of this phylum are remarkably diverse in terms of life cycle, morphology, physiology and ecology. Recent comparative genomic analysis of 19 actinobacterial species determined that only 5 genes of unknown function uniquely define this large phylum [1]. The cellular functions of these actinobacteria-specific proteins (ASP) are not known.

  7. The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases.

    PubMed

    Garbers, Christoph; Monhasery, Niloufar; Aparicio-Siegmund, Samadhi; Lokau, Juliane; Baran, Paul; Nowell, Mari A; Jones, Simon A; Rose-John, Stefan; Scheller, Jürgen

    2014-09-01

    The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.

  8. Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence.

    PubMed

    Montigny, Cédric; Penin, François; Lethias, Claire; Falson, Pierre

    2004-01-28

    Transmembrane (TM) peptides often induce toxic effects when expressed in bacteria, probably due to membrane destabilization. We report here that in the case of the TM domains of hepatitis C virus (HCV) E1 and E2 envelope proteins, which are both particularly toxic for the bacteria, the insertion of the Asp-Pro (DP) sequence dramatically reduced their toxicities and promoted their expressions when produced as glutathione S-transferase (GST) GST-DP-TM chimeras. Subcellular fractionation showed that these chimeras co-sediment with the membrane fraction and contain active GST that could be solubilized with a mild detergent. Surprisingly, immuno-gold electron microscopy clearly showed that such chimeras are not localized in the membrane but in the cytosol. We thus postulate that they likely form proteo-lipidic aggregates, which prevent the bacteria from toxicity by sequestering the TM part of the chimeras. The reduction of toxicity in the presence of the Asp-Pro sequence is possibly due to Asp's negative charge that probably disadvantages the binding of the TM peptides to the membrane. In addition, the structural features of Pro residue could promote the formation of chimera aggregates. PMID:14757220

  9. Role of Asp51 and Glu105 in the enzymatic activity of a ribonuclease from Rhizopus niveus.

    PubMed

    Ohgi, K; Horiuchi, H; Watanabe, H; Iwama, M; Takagi, M; Irie, M

    1993-02-01

    The active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and Glu105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that Glu105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.

  10. Use of procalcitonin (PCT) to guide discontinuation of antibiotic use in an unspecified sepsis is an antimicrobial stewardship program (ASP).

    PubMed

    Liew, Y X; Chlebicki, M P; Lee, W; Hsu, L Y; Kwa, A L

    2011-07-01

    Clinicians have used procalcitonin (PCT) (biomarker to differentiate bacterial from non-bacterial sepsis) to guide use of antibiotics in patients. As the data for utility of PCT to discontinue antibiotics in an antimicrobial stewardship program (ASP) are lacking, we aim to describe the outcomes of patients in whom PCT was used to discontinue antibiotics under our ASP. An antimicrobial stewardship (AS) team intervened to discontinue antibiotics in patients with persistent fever or leucocytosis, source of sepsis unknown or negative bacteriological cultures, who had completed an adequate course of antibiotic therapy and had a PCT of <0.5 μg/L. Main outcomes evaluated were 14-day re-infection, 30-day mortality and readmission. Antibiotic therapy was discontinued in 42 patients in 1 year. Unknown source of sepsis was found in 38% of the patients (including possible malignant fever) and culture-negative pneumonia was found in 21%. Two patients died of advanced cancer. One patient decided for comfort care and died one week later. One patient died due to a second episode of pneumonia 37 days after first PCT test. Six patients were readmitted within 30 days due to non-infectious causes. Three patients were readmitted due to culture-negative pneumonia. None had a 14-day re-infection. PCT used to discontinue antibiotics under our ASP did not compromise patients' outcome.

  11. Death Adder Envenoming Causes Neurotoxicity Not Reversed by Antivenom - Australian Snakebite Project (ASP-16)

    PubMed Central

    Johnston, Christopher I.; O'Leary, Margaret A.; Brown, Simon G. A.; Currie, Bart J.; Halkidis, Lambros; Whitaker, Richard; Close, Benjamin; Isbister, Geoffrey K.

    2012-01-01

    Background Death adders (Acanthophis spp) are found in Australia, Papua New Guinea and parts of eastern Indonesia. This study aimed to investigate the clinical syndrome of death adder envenoming and response to antivenom treatment. Methodology/Principal Findings Definite death adder bites were recruited from the Australian Snakebite Project (ASP) as defined by expert identification or detection of death adder venom in blood. Clinical effects and laboratory results were collected prospectively, including the time course of neurotoxicity and response to treatment. Enzyme immunoassay was used to measure venom concentrations. Twenty nine patients had definite death adder bites; median age 45 yr (5–74 yr); 25 were male. Envenoming occurred in 14 patients. Two further patients had allergic reactions without envenoming, both snake handlers with previous death adder bites. Of 14 envenomed patients, 12 developed neurotoxicity characterised by ptosis (12), diplopia (9), bulbar weakness (7), intercostal muscle weakness (2) and limb weakness (2). Intubation and mechanical ventilation were required for two patients for 17 and 83 hours. The median time to onset of neurotoxicity was 4 hours (0.5–15.5 hr). One patient bitten by a northern death adder developed myotoxicity and one patient only developed systemic symptoms without neurotoxicity. No patient developed venom induced consumption coagulopathy. Antivenom was administered to 13 patients, all receiving one vial initially. The median time for resolution of neurotoxicity post-antivenom was 21 hours (5–168). The median peak venom concentration in 13 envenomed patients with blood samples was 22 ng/mL (4.4–245 ng/mL). In eight patients where post-antivenom bloods were available, no venom was detected after one vial of antivenom. Conclusions/Significance Death adder envenoming is characterised by neurotoxicity, which is mild in most cases. One vial of death adder antivenom was sufficient to bind all circulating venom. The

  12. MPEG-4 ASP SoC receiver with novel image enhancement techniques for DAB networks

    NASA Astrophysics Data System (ADS)

    Barreto, D.; Quintana, A.; García, L.; Callicó, G. M.; Núñez, A.

    2007-05-01

    This paper presents a system for real-time video reception in low-power mobile devices using Digital Audio Broadcast (DAB) technology for transmission. A demo receiver terminal is designed into a FPGA platform using the Advanced Simple Profile (ASP) MPEG-4 standard for video decoding. In order to keep the demanding DAB requirements, the bandwidth of the encoded sequence must be drastically reduced. In this sense, prior to the MPEG-4 coding stage, a pre-processing stage is performed. It is firstly composed by a segmentation phase according to motion and texture based on the Principal Component Analysis (PCA) of the input video sequence, and secondly by a down-sampling phase, which depends on the segmentation results. As a result of the segmentation task, a set of texture and motion maps are obtained. These motion and texture maps are also included into the bit-stream as user data side-information and are therefore known to the receiver. For all bit-rates, the whole encoder/decoder system proposed in this paper exhibits higher image visual quality than the alternative encoding/decoding method, assuming equal image sizes. A complete analysis of both techniques has also been performed to provide the optimum motion and texture maps for the global system, which has been finally validated for a variety of video sequences. Additionally, an optimal HW/SW partition for the MPEG-4 decoder has been studied and implemented over a Programmable Logic Device with an embedded ARM9 processor. Simulation results show that a throughput of 15 QCIF frames per second can be achieved with low area and low power implementation.

  13. Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)

    PubMed Central

    Bruce, D; Perry, D J; Borg, J Y; Carrell, R W; Wardell, M R

    1994-01-01

    A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4 degrees C and rapidly at 41 degrees C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias. Images PMID:7989582

  14. Time-resolved titrations of the Schiff base and of the Asp85 residue in artificial bacteriorhodopsins.

    PubMed

    Druckmann, S; Ottolenghi, M; Rousso, I; Friedman, N; Sheves, M

    1995-09-19

    Deprotonation/protonation processes involving the retinal Schiff base and the Asp85 residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pKa values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp85, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pKa values of the above two residues are substantially modified: 13-CF3 bR, pKa (Schiff base) = 8.2 +/- 0.2; 13-demethyl-11,14-epoxy bR, pKa (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pKa (Asp85) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pKa (Asp85) congruent to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp85 moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Association between the XPG Asp1104His and XPF Arg415Gln Polymorphisms and Risk of Cancer: A Meta-Analysis

    PubMed Central

    Wei, Wu; Liu, Yi; Su, Jiao; Wang, Su-Lan; Shen, Xu-Liang; Yang, Xian-Bin

    2014-01-01

    Backgroud The XPG (xeroderma pigmentosum type G) Asp1104His and XPF (xeroderma pigmentosum type F) Arg415Gln polymorphisms had been implicated in cancer susceptibility. The previous published data on the association between XPG Asp1104His and XPF Arg415Gln polymorphisms and cancer risk remained controversial. Methodology/Principal Findings To derive a more precise estimation of the association between the XPG Asp1104His and XPF Arg415Gln polymorphisms and overall cancer risk, we performed a meta-analysis to investigate the association between cancer susceptibility and XPG Asp1104His (32,162 cases and 39,858 controls from 66 studies) and XPF Arg415Gln polymorphisms (17,864 cases and 20,578 controls from 32 studies) in different inheritance models. We used odds ratios with 95% confidence intervals to assess the strength of the association. Overall, significantly elevated cancer risk was found when all studies were pooled into the meta-analysis of XPG Asp1104His (dominant model: OR = 1.05, 95% CI = 1.00–1.10; Asp/His vs. Asp/Asp: OR = 1.06, 95% CI = 1.01–1.11). In the further stratified and sensitivity analyses, significantly decreased lung cancer risk was found for XPF Arg415Gln (dominant model: OR = 0.82, 95% CI = 0.71–0.96; Arg/Gln versus Arg/Arg: OR = 0.83, 95% CI = 0.71–0.97; additive model: OR = 0.83, 95% CI = 0.72–0.95) and significantly increased other cancer risk was found among hospital-based studies for XPG Asp1104His (dominant model: OR = 1.23, 95% CI = 1.02–1.49). Conclusions/Significance In summary, this meta-analysis suggests that XPF Arg415Gln polymorphism may be associated with decreased lung cancer risk and XPG Asp1104His may be a low-penetrant risk factor in some cancers development. And larger scale primary studies are required to further evaluate the interaction of XPG Asp1104His and XPF Arg415Gln polymorphisms and cancer risk in specific populations. PMID:24802942

  16. The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution.

    PubMed

    Mateja, Agnieszka; Devedjiev, Yancho; Krowarsch, Daniel; Longenecker, Kenton; Dauter, Zbigniew; Otlewski, Jacek; Derewenda, Zygmunt S

    2002-12-01

    It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the protein's stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the protein's recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.

  17. A linkage of the pK{sub a}`s of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin

    SciTech Connect

    Richter, H.T.; Brown, L.S.; Lanyi, J.K.; Needleman, R.

    1996-04-02

    Because asp-85 is the acceptor of the retinal Schiff base proton during light-driven proton transport by bacteriorhodopsin, modulation of its pK{sub a} in the photocycle is to be expected. The complex titration of asp-85 in the unphotolyzed protein was suggested to reflect the dependence of this residue on the protonation state of another, unidentified group. from the pH dependencies of the rate constant for the thermal equilibration of retinal isomeric states (dark adaptation) and the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. The nature of its interaction with our recent finding that glu-204 is the origin of the proton released to the extracellular surface upon protonation of asp-85 during that transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base shifts toward complete proton transfer. This constitutes the first phase of the reprotonation switch because it excludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the change of the access of the Schiff base toward the cytoplasmic side at the second phase of the switch, and the lowering the pK{sub a} of asp-96, so as to make it a proton donor, as the third phase. 77 refs., 6 figs.

  18. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  19. Genotype at the missense G894T polymorphism (Glu298Asp) in the NOS3 gene is associated with susceptibility to acute mountain sickness.

    PubMed

    Wang, Pei; Koehle, Michael S; Rupert, Jim L

    2009-01-01

    Acute mountain sickness (AMS) is a potentially serious affliction that frequently occurs in travelers to altitudes above 2500 m. The probability of developing AMS depends on environmental factors such as rate of ascent and altitude attained; however, familial clustering and recurrence rates suggest that there may be a genetic contribution to the etiology of the condition. The underlying pathophysiology of AMS is unknown, but it may involve vasogenic edema secondary to hypoxia-induced sympathetic response and endothelial dysfunction. Nitric oxide is a potent vasomodulator, and variants in the gene that encodes endothelial nitric oxide synthase (NOS3) have been shown to affect blood pressure. We tested the hypothesis that haplotypes, as determined by tagSNPs, in NOS3 would be differentially represented in individuals with and without AMS sampled at the Janai Purnima Festival at Lake Gosain Kunda, Nepal, at 4380 m. Seven SNPs were tested, and a highly significant association (p = 0.004) was found for genotypes of the commonly studied missense polymorphism Glu298Asp (rs 1799983; G/T transversion at base 894). The T allele, which previously has been associated with hypertension, was overrepresented in individuals with AMS (0.30 vs. 0.10), but not significantly when the data were corrected for multiple testing (p = 0.024). These data suggest that a variant in a gene involved in nitric oxide synthesis is a risk factor for developing AMS. PMID:19775216

  20. A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

    PubMed

    Guo, Jingjing; Yang, Yi; Xiao, Wenjun; Sun, Weilai; Yu, Hong; Du, Lanying; Lustigman, Sara; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-04-15

    The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use. PMID:25736195

  1. A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

    PubMed

    Guo, Jingjing; Yang, Yi; Xiao, Wenjun; Sun, Weilai; Yu, Hong; Du, Lanying; Lustigman, Sara; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-04-15

    The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use.

  2. Elucidating the Role of Many-Body Forces in Liquid Water. I. Simulations of Water Clusters on the VRT (ASP-W) Potential Surfaces

    SciTech Connect

    Goldman, N; Saykally, R J

    2003-10-03

    We test the new VRT(ASP-W)II and VRT(ASP-W)III potentials by employing Diffusion Quantum Monte Carlo simulations to calculate the vibrational ground-state properties of water clusters. These potentials are fits of the highly detailed ASP-W ab initio potential to (D{sub 2}O){sub 2} microwave and far-IR data, and along with the SAPT5s potentials, are the most accurate water dimer potential surfaces in the literature. The results from VRT(ASP-W)II and III are compare to those from the original ASP-W potential, the SAPT5s family of potentials, and several bulk water potentials. Only VRT(ASP-W)II and the spectroscopically ''tuned'' SAPT5st (with N-body induction included) accurately reproduce the vibrational ground-state structures of water clusters up to the hexamer. Finally, the importance of many-body induction and three-body dispension are examined, and it is shown that the latter can have significant effects on water cluster properties despite its small magnitude.

  3. Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

    PubMed

    Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

    2016-11-01

    A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect.

  4. Bacterial communities and enzymatic activities in the vegetation-activated sludge process (V-ASP) and related advantages by comparison with conventional constructed wetland.

    PubMed

    Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Zhao, Ke; Du, Changhang; Shao, Yunxian

    2016-11-01

    A new-developed vegetation-activated sludge process (V-ASP) was implemented for decentralized domestic wastewater treatment, and studied in lab-scale and full-scale. The main purpose of this work was the investigation of biomass activities and microbial communities in V-ASP by comparison with conventional constructed wetland (CW), to unveil the causations of its consistently higher pollutants removal efficiencies. Compared with CWs, V-ASP has greater vegetation nitrogen and phosphorus uptake rates, higher biomass and enzymatic activities, and more bacteria community diversity. The microbial community structure was comprehensively analyzed by using high-throughput sequencing. It was observed that Proteobacteria was dominated in both CWs and V-ASPs, while their subdivisions distribution was rather different. V-ASPs contained a higher nitrite-oxidizing bacteria (Nitrospira) abundances that resulted in a consistently better nitrogen removal efficiency. Hence, a long-term experiment of full-scale V-ASP displayed stably excellent capability in resistance of influent loading shocks and seasonal temperature effect. PMID:27591520

  5. Association of NOS3 Glu298Asp SNP with hypertension and possible effect modification of dietary fat intake in the ARIC study.

    PubMed

    Kingah, Pascal L; Luu, Hung N; Volcik, Kelly A; Morrison, Alanna C; Nettleton, Jennifer A; Boerwinkle, Eric

    2010-02-01

    Endothelial nitric oxide synthase breaks down nitric oxide and has a key role in blood pressure regulation. Earlier studies have shown associations between single nucleotide polymorphisms (SNPs) in the NOS3 gene and hypertension. Studies also suggest that such associations may vary by dietary fat intake. We investigated associations between the NOS3 Glu298Asp SNP (rs1799983) and hypertension, as well as the interaction between NOS3 genotypes and dietary fat intake using data from baseline examination in white and African American participants in the Atherosclerosis Risk in Community (ARIC) study. Dietary fat intake was measured by a Food Frequency Questionnaire during the baseline examination in 15 792 individuals aged 45-64 years in ARIC study participants. Race-stratified unconditional logistic regression was performed to investigate the association between prevalent hypertension and NOS3 Glu298Asp genotypes. There was no significant interaction between dietary fat intake and NOS3 Glu298Asp genotype with regards to hypertension status in either African Americans or whites (P for interaction=0.3 and 0.4, respectively). We found a significant relationship between NOS3 Glu298Asp and triglycerides in African Americans. We did not find an association between the NOS3 Glu298Asp polymorphism and hypertension, and dietary fat intake did not interact with NOS3 genotypes to influence hypertension. We recommend further exploration of the relationship between NOS3 Glu298Asp and triglycerides in African Americans. PMID:19960019

  6. Microparticles Mediate Hepatic Ischemia-Reperfusion Injury and Are the Targets of Diannexin (ASP8597)

    PubMed Central

    Wong, Heng Jian; Croft, Kevin; Mori, Trevor; Farrell, Geoffrey C.

    2014-01-01

    Background & Aims Ischemia–reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis. Methods C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min–24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function. Results Microparticles were detected in the circulation 15–30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory

  7. Three rare diseases in one Sib pair: RAI1, PCK1, GRIN2B mutations associated with Smith-Magenis Syndrome, cytosolic PEPCK deficiency and NMDA receptor glutamate insensitivity.

    PubMed

    Adams, David R; Yuan, Hongjie; Holyoak, Todd; Arajs, Katrina H; Hakimi, Parvin; Markello, Thomas C; Wolfe, Lynne A; Vilboux, Thierry; Burton, Barbara K; Fajardo, Karin Fuentes; Grahame, George; Holloman, Conisha; Sincan, Murat; Smith, Ann C M; Wells, Gordon A; Huang, Yan; Vega, Hugo; Snyder, James P; Golas, Gretchen A; Tifft, Cynthia J; Boerkoel, Cornelius F; Hanson, Richard W; Traynelis, Stephen F; Kerr, Douglas S; Gahl, William A

    2014-11-01

    The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.

  8. Three Rare Diseases in One Sib Pair: RAI1, PCK1, GRIN2B Mutations Associated with Smith-Magenis Syndrome, Cytosolic PEPCK Deficiency and NMDA Receptor Glutamate Insensitivity

    PubMed Central

    Adams, David R; Yuan, Hongjie; Holyoak, Todd; Arajs, Katrina H.; Hakimi, Parvin; Markello, Thomas C; Wolfe, Lynne A; Vilboux, Thierry; Burton, Barbara K; Fajardo, Karin Fuentes; Grahame, George; Holloman, Conisha; Sincan, Murat; Smith, Ann C M; Wells, Gordon A; Huang, Yan; Vega, Hugo; Snyder, James P; Golas, Gretchen A; Tifft, Cynthia J; Boerkoel, Cornelius F; Hanson, Richard W; Traynelis, Stephen F; Kerr, Douglas S; Gahl, William A

    2014-01-01

    The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. As demonstration of a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases. PMID:24863970

  9. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  10. Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in Heterosigma akashiwo (Raphidophyceae)

    PubMed Central

    Duplessis, Melinda R; Karol, Kenneth G; Adman, Elinor T; Choi, Lauren YS; Jacobs, Michael A; Cattolico, Rose Ann

    2007-01-01

    Background Maintenance of homeostasis requires that an organism perceive selected physical and chemical signals within an informationally dense environment. Functionally, an organism uses a variety of signal transduction arrays to amplify and convert these perceived signals into appropriate gene transcriptional responses. These changes in gene expression serve to modify selective metabolic processes and thus optimize reproductive success. Here we analyze a chloroplast-encoded His-to-Asp signal transduction circuit in the stramenopile Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor]. The presence, structure and putative function of this protein pair are discussed in the context of their evolutionary homologues. Results Bioinformatic analysis of the Heterosigma akashiwo chloroplast genome sequence revealed the presence of a single two-component His-to-Asp (designated Tsg1/Trg1) pair in this stramenopile (golden-brown alga). These data represent the first documentation of a His-to-Asp array in stramenopiles and counter previous reports suggesting that such regulatory proteins are lacking in this taxonomic cluster. Comparison of the 43 kDa H. akashiwo Tsg1 with bacterial sensor kinases showed that the algal protein exhibits a moderately maintained PAS motif in the sensor kinase domain as well as highly conserved H, N, G1 and F motifs within the histidine kinase ATP binding site. Molecular modelling of the 27 kDa H. akashiwo Trg1 regulator protein was consistent with a winged helix-turn-helix identity – a class of proteins that is known to impact gene expression at the level of transcription. The occurrence of Trg1 protein in actively growing H. akashiwo cells was verified by Western analysis. The presence of a PhoB-like RNA polymerase loop in Trg1 and its homologues in the red-algal lineage support the hypothesis that Trg1 and its homologues interact with a sigma 70 (σ70) subunit (encoded by rpoD) of a eubacterial

  11. Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad.

    PubMed

    Moynihan, Patrick J; Clarke, Anthony J

    2014-10-01

    The O-acetylation of the essential cell wall polymer peptidoglycan is essential in many bacteria for their integrity and survival, and it is catalyzed by peptidoglycan O-acetlytransferase B (PatB). Using PatB from Neisseria gonorrhoeae as the model, we have shown previously that the enzyme has specificity for polymeric muropeptides that possess tri- and tetrapeptide stems and that rates of reaction increase with increasing degrees of polymerization. Here, we present the catalytic mechanism of action of PatB, the first to be described for an O-acetyltransferase of any bacterial exopolysaccharide. The influence of pH on PatB activity was investigated, and pKa values of 6.4-6.45 and 6.25-6.35 for the enzyme-substrate complex (kcat vs pH) and the free enzyme (kcat·KM(-1) vs pH), respectively, were determined for the respective cosubstrates. The enzyme is partially inactivated by sulfonyl fluorides but not by EDTA, suggesting the participation of a serine residue in its catalytic mechanism. Alignment of the known and hypothetical PatB amino acid sequences identified Ser133, Asp302, and His305 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of Asp302 with Ala resulted in an enzyme with less than 20% residual activity, whereas activity was barely detectable with (His305 → Ala)PatB and (Ser133 → Ala)PatB was totally inactive. The reaction intermediate of the transferase reaction involving acetyl- and propionyl-acyl donors was trapped on both the wild-type and (Asp302 → Ala) enzymes and LC-MS/MS analysis of tryptic peptides identified Ser133 as the catalytic nucleophile. A transacetylase mechanism is proposed based on the mechanism of action of serine esterases. PMID:25215566

  12. A new alpha chain hemoglobin variant: Hb Al-Hammadi Riyadh [alpha75(EF4)Asp-->Val (alpha2)].

    PubMed

    Burnichon, Nelly; Lacan, Philippe; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Mowafy, Mohammed; Couprie, Nicole; Francina, Alain

    2006-01-01

    A new hemoglobin (Hb) variant in the heterozygous state, Hb Al-Hammadi Riyadh [codon 75 (GAC-->GTC); alpha75(EF4)Asp-->Val (alpha2)] corresponding to an A-->T transversion on the second exon of the alpha2-globin gene, is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). The variant was found during a routine Hb analysis for anemia in a 16-month-old boy who lived in Riyadh, Kingdom of Saudi Arabia.

  13. Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

    PubMed

    Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

    2013-09-01

    Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes.

  14. Sequence conservation in the Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) from hookworm infected individuals in Thailand.

    PubMed

    Ungcharoensuk, Charoenchai; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

    2012-12-01

    The Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) was one of the promising vaccine candidates against the most prevalent human hookworm species as adverse vaccine reaction has compromised further human vaccine trials. To elucidate the gene structure and the extent of sequence diversity, we determined the complete nucleotide sequence of the Na-asp-2 gene of individual larvae from 32 infected subjects living in 3 different endemic areas of Thailand. Sequence analysis revealed that the gene encoding Na-ASP-2 comprised 8 exons. Of 3 nucleotide substitutions in these exons, only one causes an amino acid change from leucine to methionine. A consensus conserved GT and AG at the 5' and the 3' boundaries of each intron was observed akin to those found in other eukaryotic genes. Introns of Na-asp-2 contained 23 nucleotide substitutions and 0-18 indels. The mean number of nucleotide substitutions per site (d) in introns was not significantly different from the mean number of synonymous substitutions per synonymous site (d(S)) in exons whereas d in introns was significantly exceeded d(N) (the mean number of nonsynonymous substitutions per nonsynonymous site) in exons (p<0.05), suggesting that introns and synonymous sites in exons may evolve at a similar rate whereas functional constraints at the amino acid could limit amino acid substitutions in Na-ASP-2. A recombination site was identified in an intron near the 3' portion of the gene. The positions of introns and the intron phases in the Na-asp-2 gene comparing with those in other pathogenesis-related-1 proteins of Loa loa, Onchocerca volvulus, Heterodera glycines, Caenorhabditis elegans and human were relatively conserved, suggesting evolutionary conservation of these genes. Sequence conservation in Na-ASP-2 may not compromise further vaccine design if adverse vaccine effects could be resolved whereas microheterogeneity in introns of this locus may be useful for population genetics analysis of N. americanus

  15. Sequence conservation in the Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) from hookworm infected individuals in Thailand.

    PubMed

    Ungcharoensuk, Charoenchai; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

    2012-12-01

    The Ancylostoma secreted protein-2 of Necator americanus (Na-ASP-2) was one of the promising vaccine candidates against the most prevalent human hookworm species as adverse vaccine reaction has compromised further human vaccine trials. To elucidate the gene structure and the extent of sequence diversity, we determined the complete nucleotide sequence of the Na-asp-2 gene of individual larvae from 32 infected subjects living in 3 different endemic areas of Thailand. Sequence analysis revealed that the gene encoding Na-ASP-2 comprised 8 exons. Of 3 nucleotide substitutions in these exons, only one causes an amino acid change from leucine to methionine. A consensus conserved GT and AG at the 5' and the 3' boundaries of each intron was observed akin to those found in other eukaryotic genes. Introns of Na-asp-2 contained 23 nucleotide substitutions and 0-18 indels. The mean number of nucleotide substitutions per site (d) in introns was not significantly different from the mean number of synonymous substitutions per synonymous site (d(S)) in exons whereas d in introns was significantly exceeded d(N) (the mean number of nonsynonymous substitutions per nonsynonymous site) in exons (p<0.05), suggesting that introns and synonymous sites in exons may evolve at a similar rate whereas functional constraints at the amino acid could limit amino acid substitutions in Na-ASP-2. A recombination site was identified in an intron near the 3' portion of the gene. The positions of introns and the intron phases in the Na-asp-2 gene comparing with those in other pathogenesis-related-1 proteins of Loa loa, Onchocerca volvulus, Heterodera glycines, Caenorhabditis elegans and human were relatively conserved, suggesting evolutionary conservation of these genes. Sequence conservation in Na-ASP-2 may not compromise further vaccine design if adverse vaccine effects could be resolved whereas microheterogeneity in introns of this locus may be useful for population genetics analysis of N. americanus.

  16. Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

    PubMed

    Acuña, Mariana; Martínez, Pablo; Moraga, Carol; He, Xingxuan; Moraga, Mauricio; Hunter, Bessie; Nuernberg, Peter; Gutiérrez, Rodrigo A; González, Mauricio; Schuchman, Edward H; Santos, José Luis; Miquel, Juan Francisco; Mabe, Paulina; Zanlungo, Silvana

    2016-02-01

    Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients.

  17. Epidemiological, clinical and biochemical characterization of the p.(Ala359Asp) SMPD1 variant causing Niemann-Pick disease type B.

    PubMed

    Acuña, Mariana; Martínez, Pablo; Moraga, Carol; He, Xingxuan; Moraga, Mauricio; Hunter, Bessie; Nuernberg, Peter; Gutiérrez, Rodrigo A; González, Mauricio; Schuchman, Edward H; Santos, José Luis; Miquel, Juan Francisco; Mabe, Paulina; Zanlungo, Silvana

    2016-02-01

    Niemann-Pick disease type B (NPDB) is a rare, inherited lysosomal storage disorder that occurs due to variants in the sphingomyelin phosphodiesterase 1 (SMPD1) gene and the resultant deficiency of acid sphingomyelinase (ASM) activity. While numerous variants causing NPDB have been described, only a small number have been studied in any detail. Herein, we describe the frequency of the p.(Ala359Asp) variant in the healthy Chilean population, and determine the haplotype background of homozygous patients to establish if this variant originated from a common founder. Genomic DNA samples from 1691 healthy individuals were analyzed for the p.(Ala359Asp) variant. The frequency of p.(Ala359Asp) was found to be 1/105.7, predicting a disease incidence of 1/44 960 in Chile, higher than the incidence estimated by the number of confirmed NPDB cases. We also describe the clinical characteristics of 13 patients homozygous for p.(Ala359Asp) and all of them had moderate to severe NPDB disease. In addition, a conserved haplotype and shared 280 Kb region around the SMPD1 gene was observed in the patients analyzed, indicating that the variant originated from a common ancestor. The haplotype frequency and mitochondrial DNA analysis suggest an Amerindian origin for the variant. To assess the effect of the p.(Ala359Asp) variant, we transfected cells with the ASM-p.(Ala359Asp) cDNA and the activity was only 4.2% compared with the wild-type cDNA, definitively demonstrating the causative effect of the variant on ASM function. Information on common variants such as p.(Ala359Asp) is essential to guide the successful implementation for future therapies and benefit to patients. PMID:25920558

  18. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD. PMID:24619194

  19. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

  20. Solute transport in crystalline rocks at Aspö--I: geological basis and model calibration.

    PubMed

    Mazurek, Martin; Jakob, Andreas; Bossart, Paul

    2003-03-01

    Water-conducting faults and fractures were studied in the granite-hosted Aspö Hard Rock Laboratory (SE Sweden). On a scale of decametres and larger, steeply dipping faults dominate and contain a variety of different fault rocks (mylonites, cataclasites, fault gouges). On a smaller scale, somewhat less regular fracture patterns were found. Conceptual models of the fault and fracture geometries and of the properties of rock types adjacent to fractures were derived and used as input for the modelling of in situ dipole tracer tests that were conducted in the framework of the Tracer Retention Understanding Experiment (TRUE-1) on a scale of metres. After the identification of all relevant transport and retardation processes, blind predictions of the breakthroughs of conservative to moderately sorbing tracers were calculated and then compared with the experimental data. This paper provides the geological basis and model calibration, while the predictive and inverse modelling work is the topic of the companion paper [J. Contam. Hydrol. 61 (2003) 175]. The TRUE-1 experimental volume is highly fractured and contains the same types of fault rocks and alterations as on the decametric scale. The experimental flow field was modelled on the basis of a 2D-streamtube formalism with an underlying homogeneous and isotropic transmissivity field. Tracer transport was modelled using the dual porosity medium approach, which is linked to the flow model by the flow porosity. Given the substantial pumping rates in the extraction borehole, the transport domain has a maximum width of a few centimetres only. It is concluded that both the uncertainty with regard to the length of individual fractures and the detailed geometry of the network along the flowpath between injection and extraction boreholes are not critical because flow is largely one-dimensional, whether through a single fracture or a network. Process identification and model calibration were based on a single uranine breakthrough

  1. Exploring the molecular basis for selective binding of Mycobacterium tuberculosis Asp kinase toward its natural substrates and feedback inhibitors: a docking and molecular dynamics study.

    PubMed

    Chaitanya, M; Babajan, B; Anuradha, C M; Naveen, M; Rajasekhar, C; Madhusudana, P; Kumar, Chitta Suresh

    2010-08-01

    Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. In this context, aspartokinase of mycobacterium tuberculosis has drawn attention for designing novel anti-TB drugs. Asp kinase is an enzyme responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate and involved in the branched biosynthetic pathway leading to the synthesis of amino acids lysine, threonine, methionine and isoleucine. An intermediate of lysine biosynthetic branch, mesodiaminopimelate is also a component of the peptidoglycan which is a component of bacterial cell wall. To interfere with the production of all these amino acids and cell wall, it is possible to inhibit Asp kinase activity. This can be achieved using Asp kinase inhibitors. In order to design novel Asp kinase inhibitors as effective anti-TB drugs, it is necessary to have an understanding of the binding sites of Asp kinase. As no crystal structure of the enzyme has yet been published, we built a homology model of Asp kinase using the crystallized Asp kinase from M. Jannaschii, as template structures (2HMF and 3C1M). After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The results of molecular docking studies with natural substrates, products and feedback inhibitors are in agreement with the published data and showed that ACT domain plays an important role in binding to ligands. Based on the docking conformations, pharmacophore model can be developed by probing the common features of ligands. By analyzing the results, ACT domain architecture, certain key residues that are responsible for binding to feedback inhibitors and natural substrates were identified. This would be very helpful in understanding the blockade mechanism of Asp kinase and providing insights

  2. NPM/ALK mutants resistant to ASP3026 display variable sensitivity to alternative ALK inhibitors but succumb to the novel compound PF-06463922.

    PubMed

    Mologni, Luca; Ceccon, Monica; Pirola, Alessandra; Chiriano, Gianpaolo; Piazza, Rocco; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2015-03-20

    ALK is involved in the onset of several tumors. Crizotinib (XalkoriTM), a potent ALK inhibitor, represents the current front-line treatment for ALK+ NSCLC and shows great clinical efficacy. However, resistant disease often develops after initial response. ASP3026 is a novel second-generation ALK inhibitor with activity on crizotinib-resistant ALK-L1196M gatekeeper mutant. As resistance is likely to be a relevant hurdle for any drug, we sought to determine the resistance profile of ASP3026 in the context of NPM/ALK+ ALCL. We selected six ASP3026-resistant cell lines by culturing human ALCL cells in the presence of increasing concentrations of drug. The established resistant cell lines carry several point mutations in the ALK kinase domain (G1128S, C1156F, I1171N/T, F1174I, N1178H, E1210K and C1156F/D1203N were the most frequent) that are shown to confer resistance to ASP3026 in the Ba/F3 cell model. All mutants were profiled for cross-resistance against a panel of clinically relevant inhibitors including ceritinib, alectinib, crizotinib, AP26113 and PF-06463922. Finally, a genetically heterogeneous ASP3026-resistant cell line was exposed to second-line treatment simulations with all inhibitors. The population evolved according to relative sensitivity of its mutant subclones to the various drugs. Compound PF-06463922 did not allow the outgrowth of any resistant clone, at non-toxic doses.

  3. Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

    PubMed

    Nakada, Naoyuki; Oda, Kazuo

    2015-01-01

    1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

  4. Capillary electrophoretic study of the degradation pathways and kinetics of the aspartyl model tetrapeptide Gly-Phe-Asp-GlyOH in alkaline solution.

    PubMed

    Brückner, Christin; Imhof, Diana; Scriba, Gerhard K E

    2013-03-25

    The aim of the present study was the investigation of the isomerization and epimerization kinetics of the aspartyl tetrapeptide Gly-Phe-Asp-GlyOH at alkaline conditions. Incubations of the model tetrapeptide in sodium borate buffer, pH 10 and ionic strength 0.2M, at 25°C and 80°C were analyzed by a validated CE-UV assay and fitted according to a pharmacokinetic model. CE-ESI-MS was used for peptide identification. Enantiomerization and isomerization of the aspartyl residue of the model tetrapeptide was observed under all experimental conditions applied. Differences in the velocity and the ratios of the rates of the degradation reactions indicated different effects of temperature on the individual reactions. At 80°C, a rapid formation of β-Asp and d-Asp containing isomers from Gly-l-Phe-α-l-Asp-GlyOH was monitored. Rate constants of the hydrolysis of the succinimide (Asu) intermediate generally exceeded the formation of the intermediate from α/β-Asp peptides. A higher rate constant was observed for the enantiomerization from l-configured Asu compared to d-Asu. At 25°C, epimerization and isomerization equilibrium was not reached within 5208h. Compared to 80°C different ratios of the individual reaction rates were noted. Moreover, inversion of the sequence of the first 2 amino acids was noted as a minor side reaction at 80°C.

  5. Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A{sub 2} with low catalytic activity from Bothrops jararacussu venom

    SciTech Connect

    Corrêa, L. C.; Marchi-Salvador, D. P.; Cintra, A. C. O.; Soares, A. M.

    2006-08-01

    A myotoxic Asp49-PLA{sub 2} with low catalytic activity from B. jararacussu (BthTX-II) was crystallized in the monoclinic crystal system; a complete X-ray diffraction data set was collected and a molecular-replacement solution was obtained. The oligomeric structure of BthTX-II resembles those of the Asp49-PLA{sub 2} PrTX-III and all bothropic Lys49-PLA{sub 2}s. For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A{sub 2} (Asp49-PLA{sub 2}) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA{sub 2} PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA{sub 2}s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA{sub 2} from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA{sub 2}s.

  6. Triple-bond reactivity of an AsP complex intermediate: synthesis stemming from molecular arsenic, As(4).

    PubMed

    Spinney, Heather A; Piro, Nicholas A; Cummins, Christopher C

    2009-11-11

    While P(4) is the stable molecular form of phosphorus, a recent study illustrated the possibility of P(2) generation for reactions in organic media under mild conditions. The heavier group 15 element arsenic can exist as As(4) molecules, but As(4) cannot be stored as a pure substance because it is both light-sensitive and reverts thermally to its stable, metallic gray form. Herein we report As(4) activation giving rise to a mu-As(2) diniobium complex, serving in turn as precursor to a terminal arsenide anion complex of niobium. Functionalization of the latter provides the new AsPNMes* ligand, which when complexed with tungsten pentacarbonyl elicits extrusion of the (AsP)W(CO)(5) molecule as a reactive intermediate. Trapping reactions of the latter with organic dienes are found to furnish double Diels-Alder adducts in which the AsP unit is embedded in a polycyclic organic framework. Thermal generation of (AsP)W(CO)(5) in the presence of the neutral terminal phosphide complex P identical withMo(N[(i)Pr]Ar)(3) leads to the cyclo-AsP(2) complex (OC)(5)W(cyclo-AsP(2))Mo(N[(i)Pr]Ar)(3). The (AsP)W(CO)(5) trapping products were crystallized and characterized by X-ray diffraction methods, and computational methods were applied for analysis of the As-As and As-P bonds in the complexes.

  7. GC Glu416Asp and Thr420Lys polymorphisms contribute to gastrointestinal cancer susceptibility in a Chinese population

    PubMed Central

    Zhou, Liqing; Zhang, Xiaojiao; Chen, Xuechao; Liu, Li; Lu, Chao; Tang, Xiaohu; Shi, Juan; Li, Meng; Zhou, Mo; Zhang, Zhouwei; Xiao, Lingchen; Yang, Ming

    2012-01-01

    Vitamin D has potent anticancer properties, especially against gastrointestinal cancers. Group-specific component (GC), a key member of vitamin D pathway proteins, could bind to and transport vitamin D to target organs. As a polymorphic protein, two common coding single nucleotide polymorphisms (SNP) [Glu416Asp (rs7041) and Thr420Lys (rs4588)] were identified in its gene. These SNPs have been associated to circulating vitamin D levels and several cancer risks in different populations. However, there is no report on their role in gastrointestinal cancer development among Chinese to date. Therefore, we examined the association between these variants and risk of gastrointestinal cancers in a case-control cohort including 964 patients with four gastrointestinal cancers (hepatocellular carcinoma, esophageal cancer, gastric cancer and colorectal cancer) and 1187 controls. Odds ratios and 95% confidence intervals were estimated by logistic regression. We found that GC Thr420Lys polymorphism has significant impact on the risk of developing gastrointestinal cancers, especially colorectal cancer. Additionally, subjects who carrying GC Asp416-Lys420 haplotype, which contains the at-risk 420Lys allele, also showed significantly increased risk to develop gastrointestinal cancers. In conclusion, our study demonstrated that common genetic variants and haplo-types in GC may influence individual susceptibility to gastrointestinal cancers in Chinese population. PMID:22328951

  8. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    PubMed Central

    Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

    2016-01-01

    Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

  9. A novel Mutein of TNFα Containing the Arg-Gly-Asp Sequence Shows Reduced Toxicity in Intestine

    PubMed Central

    Miyata, K.; Sakae, N.; Kuroda, K.; Nishimura, K.; Yotsuya, S.; Kato, M.

    1994-01-01

    The effects of human tumour necrosis factor-α (TNFα), or its mutein (F4168) having the cell adhesive Arg-Gly-Asp sequence at the N-terminus, on intestinal injury, were examined. Histopathological examination revealed that an intravenous injection of TNFα resulted in marked haemorrhage or oedema in the caecum of rats, whereas F4168 showed no such effects even at the same therapeutic dose. Moreover, the number of neutrophils that adhered to endothelial cells or infiltrated the mucosal tissue was much higher after TNFα injection compared with F4168 in vivo. The enhanced adhesion of neutrophils on to human umbilical vein endothelial cells also occurred when the latter were pre-stimulated with TNFα but not with F4168 in vitro. The expression of the cell adhesion molecules including endothelial leukocyte adhesion molecule-1 or intercellular adhesion molecule-1 on F4168- stimulated human umbilical vein endothelial ceils was significantly lower than that stimulated with TNFα. These results suggest that the Arg-Gly-Asp sequence introduced into the TNFα molecule abrogates the side effect of this cytokine such as tissue injury or shock, and that F4168 could be useful for systemic therapy. PMID:18472928

  10. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  11. Enhancing the Activity of Peptide-Based Artificial Hydrolase with Catalytic Ser/His/Asp Triad and Molecular Imprinting.

    PubMed

    Wang, Mengfan; Lv, Yuqi; Liu, Xiaojing; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-06-01

    In this study, an artificial hydrolase was developed by combining the catalytic Ser/His/Asp triad with N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF), followed by coassembly of the peptides into nanofibers (CoA-HSD). The peptide-based nanofibers provide an ideal supramolecular framework to support the functional groups. Compared with the self-assembled catalytic nanofibers (SA-H), which contain only the catalytic histidine residue, the highest activity of CoA-HSD occurs when histidine, serine, and aspartate residues are at a ratio of 40:1:1. This indicates that the well-ordered nanofiber structure and the synergistic effects of serine and aspartate residues contribute to the enhancement in activity. Additionally, for the first time, molecular imprinting was applied to further enhance the activity of the peptide-based artificial enzyme (CoA-HSD). p-NPA was used as the molecular template to arrange the catalytic Ser/His/Asp triad residues in the proper orientation. As a result, the activity of imprinted coassembled CoA-HSD nanofibers is 7.86 times greater than that of nonimprinted CoA-HSD and 13.48 times that of SA-H.

  12. Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant.

    PubMed

    Zhao, Guangyu; Du, Lanying; Xiao, Wenjun; Sun, Shihui; Lin, Yongping; Chen, Min; Kou, Zhihua; He, Yuxian; Lustigman, Sara; Jiang, Shibo; Zheng, Bo-Jian; Zhou, Yusen

    2010-10-18

    Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions.

  13. Crystal structure of the extracellular segment of integrin {alpha}V{beta}3 in complex with an Arg-Gly-Asp ligand.

    SciTech Connect

    Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Goodman, S.; Arnaout, M. A.; Biosciences Division; Massachusetts General Hospital; Harvard Medical School

    2002-04-05

    The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.

  14. ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

    PubMed Central

    Yamamoto, Rie; Okada, Youhei; Hirose, Jun; Koshika, Tadatsura; Kawato, Yuka; Maeda, Masashi; Saito, Rika; Hattori, Kazuyuki; Harada, Hironori; Nagasaka, Yasuhisa; Morokata, Tatsuaki

    2014-01-01

    Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1–S1P5). S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod. PMID:25347187

  15. The apolipoprotein epsilon4 allele confers additional risk in children with familial hypercholesterolemia.

    PubMed

    Wiegman, Albert; Sijbrands, Eric J G; Rodenburg, Jessica; Defesche, Joep C; de Jongh, Saskia; Bakker, Henk D; Kastelein, John J P

    2003-06-01

    Children with familial hypercholesterolemia (FH) exhibit substantial variance of LDL cholesterol. In previous studies, family members of children with FH were included, which may have influenced results. To avoid such bias, we studied phenotype in 450 unrelated children with FH and in 154 affected sib-pairs. In known families with classical FH, diagnosis was based on plasma LDL cholesterol above the age- and gender-specific 95th percentile. Girls had 0.47 +/- 0.15 mmol/L higher LDL cholesterol, compared with boys (p = 0.002). Also in girls, HDL cholesterol increased by 0.07 +/- 0.03 mmol/L per 5 y (pfor trend = 0.005); this age effect was not observed in boys. The distribution of apolipoprotein (apo) E genotypes was not significantly different between probands, their paired affected siblings, or a Dutch control population. Carriers with or without one epsilon4 allele had similar LDL and HDL cholesterol levels. Within the affected sib-pairs, the epsilon4 allele explained 72.4% of the variance of HDL cholesterol levels (-0.15 mmol/L, 95% confidence interval -0.24 to -0.05, p = 0.003). The effect of apoE4 on HDL cholesterol differed with an analysis based on probands or on affected sib-pairs. The affected sib-pair model used adjustment for shared environment, type of LDL receptor gene mutation, and a proportion of additional genetic factors and may, therefore, be more accurate in estimating effects of risk factors on complex traits. We conclude that the epsilon4 allele was associated with lower HDL cholesterol levels in an affected sib-pair analysis, which strongly suggests that apoE4 influences HDL cholesterol levels in FH children. Moreover, the strong association suggests that apoE4 carries an additional disadvantage for FH children. PMID:12646733

  16. Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.

    PubMed

    Vargas-Muñiz, José M; Renshaw, Hilary; Richards, Amber D; Waitt, Greg; Soderblom, Erik J; Moseley, Martin A; Asfaw, Yohannes; Juvvadi, Praveen R; Steinbach, William J

    2016-01-01

    Septins are a conserved family of GTPases that form hetero-oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspB (mt) -T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A

  17. Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus

    PubMed Central

    Vargas-Muñiz, José M.; Renshaw, Hilary; Richards, Amber D.; Waitt, Greg; Soderblom, Erik J.; Moseley, Martin. A.; Asfaw, Yohannes; Juvvadi, Praveen R.; Steinbach, William J.

    2016-01-01

    Septins are a conserved family of GTPases that form hetero–oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspBmt-T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A

  18. Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.

    PubMed

    Vargas-Muñiz, José M; Renshaw, Hilary; Richards, Amber D; Waitt, Greg; Soderblom, Erik J; Moseley, Martin A; Asfaw, Yohannes; Juvvadi, Praveen R; Steinbach, William J

    2016-01-01

    Septins are a conserved family of GTPases that form hetero-oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspB (mt) -T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A

  19. Adrenocorticotropin receptor/melanocortin receptor-2 maps within a reported susceptibility region for bipolar illness on chromosome 18

    SciTech Connect

    Detera-Wadleigh, S.D.; Yoon, Sung W.; Goldin, L.R.

    1995-08-14

    We have examined the possible linkage of adrenocorticotropin receptor/melanocortin receptor-2 (ACTHR/MC-2) to a reported putative susceptibility locus for bipolar illness (BP) in 20 affected pedigrees. Initially, allelic variants of the gene were identified by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) and the gene was genetically mapped using both the Centre d`Etudes du Polymorphisme Humain (CEPH) pedigrees and the BP pedigrees used in this study. We found that the ACTHR/MC-2 gene maps between D18S53 and D18S66. These loci span a region of chromosome 18 which, in a previous study revealed a putative predisposing locus to BP through nonparametric methods of analyses, although affected sib-pair (ASP) method revealed an increase in allele sharing among ill individuals, P=0.023. Since this receptor is within a potential linkage region, ACTHR/MC-2 could be considered a candidate gene for BP. 22 refs., 4 figs., 2 tabs.

  20. Recombinant C3adesArg/acylation stimulating protein (ASP) is highly bioactive: a critical evaluation of C5L2 binding and 3T3-L1 adipocyte activation.

    PubMed

    Cui, Wei; Lapointe, Marc; Gauvreau, Danny; Kalant, David; Cianflone, Katherine

    2009-10-01

    C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (Fl-ASP) binding to C5L2 increased from transiently transfectedASP-sorted C5L2-HEK for both human C5L2 and mouse C5L2. Transfected C5L2-CHO cells had similar results. Endogenous C5L2 expression increased from 3T3-L1 preadipocytes<3T3-L1 adipocytesASP demonstrated background fluorescence only. In adherent C5L2-HEK (Fl-ASP sorted) and 3T3-L1 cells, blocking with 10% fetal calf serum, protamine sulfate or ovalbumin prevented (125)I-ASP non-specific binding (NSB, no cells), while albumin increased NSB. Binding to non-transfected HEK was comparable to NSB. Optimal specific binding was obtained at 20 degrees C (vs. 4 degrees C) in PBS or serum-free medium with K(d) 83.7+/-23.7 nM (C5L2-HEK), 66+/-15 nM (C5L2-CHO) and 76+/-14.3 nM (3T3-L1 preadipocytes); (125)I-C5a binding had greater affinity. Fl-ASP-C5L2 binding was comparable and concentration dependent (K(d) 31 nM (direct binding) and IC(50) 35 nM (competition binding) regardless of conditions). Recombinant ASP (rASP) produced in modified Escherichia coli Origami (DE3) (allowing folding and disulphide bridge formation), purified under non-denaturing conditions demonstrated 10x greater bioactivity vs. proteolytically derived plasma ASP for triglyceride synthesis and fatty acid uptake in 3T3-L1 adipocytes and preadipocytes while adipose tissue from C5L2 KO mice was non-responsive. rASP stimulation of adipocyte BODIPY-fatty acid uptake demonstrated EC(50) 115+/-93 nM and maximal stimulation of 413+/-33%, p<0.001. ASP binding has distinct characteristics that lead to C5L2 activation and increased

  1. Affective Learning.

    ERIC Educational Resources Information Center

    Brown, Charles T.

    This paper addresses itself to the question, "What does feeling have to do with knowing?" Two movements in affective education are discussed which have come into focus in recent years and which attempt to define the relationship between knowing and feeling. The first, a conscious application of the role of arousal in learning, emphasizes arousal…

  2. Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

    PubMed Central

    Sucasaca-Monzón, Georgina; Randazzo-Moura, Priscila; Rocha, Thalita; Vilca-Quispe, Augusto; Ponce-Soto, Luis Alberto; Marangoni, Sérgio; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa

    2015-01-01

    A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom. PMID:25789175

  3. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

    SciTech Connect

    Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi; Yam, Katherine C.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2013-11-12

    The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ESred, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ESred decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ESred represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

  4. Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

    PubMed

    McIntosh, D B; Woolley, D G; MacLennan, D H; Vilsen, B; Andersen, J P

    1999-09-01

    The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.

  5. Availability of type II diabetic families for detection of diabetes susceptibility genes.

    PubMed

    Cook, J T; Page, R C; O'Rahilly, S; Levy, J; Holman, R; Barrow, B; Hattersley, A T; Shaw, A G; Wainscoat, J S; Turner, R C

    1993-10-01

    Type II diabetes is a familial disorder, as evidenced by the increased prevalence in monozygotic cotwins and first-degree relatives of affected subjects; however, its genetic etiology is largely unknown. Well-characterized pedigrees are an essential resource for the study of susceptibility genes for type II diabetes. This study describes a 5-yr search for type II diabetic families in Oxfordshire, U.K. We interviewed 950 type II diabetic subjects concerning the availability of first-degree relatives; 127 Caucasian families ascertained through a proband with type II diabetes were studied, and 589 first-degree relatives were characterized. Three large pedigrees with maturity-onset diabetes of the young, and 8 multiplex multigenerational type II diabetic pedigrees were identified. We identified 12 sib-pairs in which both siblings had type II diabetes; however, only 7 sib-pairs had both parents alive, and 2 of these had both parents affected. If one also considers one sib having diabetes and one sib having glucose intolerance as being an affected sib-pair, we identified 30 sib-pairs of which 7 had both parents affected and probably had bilineal inheritance. We identified 76 complete nuclear families with both parents and offspring available for study, but only 6 were of optimal structure for linkage analysis. In conclusion, multiplex pedigrees and type II diabetic sib-pairs with living parents are uncommon, and their ascertainment requires a substantial investment of resources. Large-scale collaborative multicenter initiatives would be needed to collect a large resource of family material for the study of susceptibility genes for type II diabetes.

  6. Adoptive immunity mediated by HLA-A*0201 restricted Asp f16 peptides-specific CD8+ T cells against Aspergillus fumigatus infection.

    PubMed

    Sun, Z; Zhu, P; Li, L; Wan, Z; Zhao, Z; Li, R

    2012-11-01

    Aspergillus fumigatus (A. fumigatus) is the most common pathogen of invasive aspergillosis (IA), a life-threatening infection in immunocompromised patients. Recent findings revealed that CD8+ T cells can mediate cytotoxic activity against A. fumigatus. Here, we bioinformatically identified three HLA-A*0201-restricted peptides from Asp f16, an A. fumigatus antigen which was previously shown to be involved in T cell immunity. Our immunological results demonstrated that these peptides can potently induce cytotoxic T lymphocyte (CTL) response in CD8+ T cells, thus, damaging the conidia and hyphae of A. fumigatus. Moreover, the Asp f16 peptides can also raise Th1 cell-like response, as measured by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT). Furthermore, we established an invasive pulmonary aspergillosis model in HLA-A*0201 transgenic mice. Adoptive transfer of Asp f16 peptides-specific CTL significantly extended the overall survival time in the A. fumigatus-infected immunocompromised mice. In conclusion, our results demonstrate that the Asp f16 peptides might provide immunity against invasive A. fumigatus infection.

  7. Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum

    PubMed Central

    Vila-Aiub, Martin M.; Yu, Qin; Han, Heping; Powles, Stephen B.

    2015-01-01

    The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. PMID:26019257

  8. XPD Asp312Asn polymorphism and esophageal cancer risk: an update meta-analysis based on 3928 cases and 6012 controls

    PubMed Central

    Guo, Xu-Feng; Wang, Jun; Lei, Xiao-Fei; Zeng, Yan-Ping; Lv, Xiao-Guang; Dong, Wei-Guo

    2014-01-01

    Background: Although xeroderma pigmentosum group D (XPD) was reported to be related with esophageal cancer (EC) risk, the results remained inconsistent. The aim of this meta-analysis was to make a more precise estimation of the relationship between XPD Asp312Asn polymorphism and EC risk. Methods: We searched PubMed, Web of Science, Embase, Medline, CNKI and Chinese Biomedical database, covering all publications (up to May, 2014). Statistical analyses were performed with Stata software (version 12.0, USA) and RevMan 5.1 (Copenhagen, 2008). The calculation of odds ratios (ORs) with 95% confidence intervals (CI) was calculated to assess the strength of the association. Results: A total of 15 case-control studies from 13 literatures including 3928 cases and 6012 controls described Asp312Asn genotypes and EC risk. A significant association between XPD Asp312Asn polymorphism and EC risk was found when all the eligible studies were pooled into this meta-analysis. It’s also the same result in subgroup analysis of smokers in dominant model (OR=1.63, 95% CI: 1.06-2.50, P=0.03). However, in the stratified analysis by ethnicity and source of population controls, no association between them was discovered. Conclusion: The XPD Asp312Asn polymorphism was proved to contribute to the risk of EC in this meta-analysis. Data showed that tobacco consumption may increase the susceptibility of EC. PMID:25356096

  9. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    PubMed

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution. PMID:26974869

  10. A novel m.7539C>T point mutation in the mt-tRNA(Asp) gene associated with multisystemic mitochondrial disease.

    PubMed

    Lehmann, Diana; Schubert, Kathrin; Joshi, Pushpa R; Baty, Karen; Blakely, Emma L; Zierz, Stephan; Taylor, Robert W; Deschauer, Marcus

    2015-01-01

    Mitochondrial transfer RNA (mt-tRNA) mutations are the commonest sub-type of mitochondrial (mtDNA) mutations associated with human disease. We report a patient with multisytemic disease characterised by myopathy, spinal ataxia, sensorineural hearing loss, cataract and cognitive impairment in whom a novel m.7539C>T mt-tRNA(Asp) transition was identified. Muscle biopsy revealed extensive histopathological findings including cytochrome c oxidase (COX)-deficient fibres. Pyrosequencing confirmed mtDNA heteroplasmy for the mutation whilst single muscle fibre segregation studies revealed statistically significant higher mutation loads in COX-deficient fibres than in COX-positive fibres. Absence from control databases, hierarchical mt-tRNA mutation segregation within tissues, and occurrence at conserved sequence positions, further confirm this novel mt-tRNA mutation to be pathogenic. To date only three mt-tRNA(Asp) gene mutations have been described with clear evidence of pathogenicity. The novel m.7539C>T mt-tRNA(Asp) gene mutation extends the spectrum of pathogenic mutations in this gene, further supporting the notion that mt-tRNA(Asp) gene mutations are associated with multisystemic disease presentations.

  11. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    PubMed

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.

  12. Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum.

    PubMed

    Vila-Aiub, Martin M; Yu, Qin; Han, Heping; Powles, Stephen B

    2015-08-01

    The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed.

  13. Molecular defects in the factor X gene caused by novel heterozygous mutations IVS5+1G>A and Asp409del.

    PubMed

    Zhou, J W; Liang, Q; Chen, Q; Xie, Y; Ding, Q L; Wang, X F; Xi, X D; Wang, H L

    2013-01-01

    Factor X (FX) deficiency is a rare autosomal-recessive bleeding disorder caused by diverse mutations in the F10 gene. To investigate the molecular basis of severe FX deficiency in a mildly hemorrhagic patient, variants of the F10 gene were detected by sequencing. A missense mutation was analysed by in vitro expression and modelling analysis, and a splice mutation using ectopic transcript analysis. The levels of activity of FX (FX:C) were <1% in both intrinsic and extrinsic pathway assays and 1.71% in chromogenic assay, the level of FX antigen (FX:Ag) was 53.36% in the proband. Two novel heterozygous mutations (IVS5+1G>A and Asp409del) were identified in the F10 gene. Ectopic transcript expression combined with informative marker (heterozygous Asp409del) analysis of the splice mutation (IVS5+1G>A) revealed and confirmed that the transcript from the mutated allele was absent, likely caused by the nonsense-mediated mRNA decay pathway. In vitro expression analysis showed that the Asp409del mutant led to a loss of enzymatic activity rather than impaired expression. Molecular modelling analysis confirmed that the Asp409del mutant dramatically altered the conformation of the 185-189 loop and impaired binding of the loop to sodium ions (Na(+) ), diminishing the enzymatic activity of FXa. This is the first report to clarify the molecular mechanisms of two naturally occurring F10 gene variants that cause severe FX deficiency.

  14. Pelvic MRI in a 17-year-old XY girl with 5-alpha reductase deficiency and a homozygous Gly115Asp mutation in SRD5A2.

    PubMed

    Sarfati, J; Trabado, S; Rocher, L; Mallet, D; Betari-Tabet, B; Morel, Y; Young, J

    2011-09-01

    We describe, here, the case report and detailed pelvic magnetic resonance imaging (MRI) aspect of a 17-year-old female XY teenager in which 5-α-reductase deficiency was caused by the homozygous Gly115Asp lost of function SRD5A2 mutation.

  15. XPC Ala499Val and XPG Asp1104His polymorphisms and digestive system cancer risk: a meta-analysis based on model-free approach

    PubMed Central

    Yu, Guangsheng; Wang, Jianlu; Dong, Jiahong; Liu, Jun

    2015-01-01

    Many studies have reported the association between XPC Ala499Val and XPG Asp1104His polymorphisms and digestive system cancer susceptibility, but the results were inconclusive. We performed a meta-analysis, using a comprehensive strategy based on the allele model and a model-free approach, to derive a more precise estimation of the relationship between XPC Ala499Val and XPG Asp1104His polymorphisms with digestive system cancer risk. For XPC Ala499Val, no significant cancer risk was found in the allele model (OR = 0.98, 95% CI: 0.86-1.11) and with model-free approach (ORG = 0.97, 95% CI: 0.83-1.13). For XPG Asp1104His, there was also no association between this polymorphism and cancer risk in the allele model (OR = 1.03, 95% CI: 0.96-1.11) and with the model-free approach (ORG = 1.04, 95% CI: 0.95-1.14). Therefore, this meta-analysis suggests that the XPC Ala499Val and XPG Asp1104His polymorphisms were not associated with digestive system cancer risk. Further large and well-designed studies are needed to confirm these findings. PMID:26131294

  16. XPC Ala499Val and XPG Asp1104His polymorphisms and digestive system cancer risk: a meta-analysis based on model-free approach.

    PubMed

    Yu, Guangsheng; Wang, Jianlu; Dong, Jiahong; Liu, Jun

    2015-01-01

    Many studies have reported the association between XPC Ala499Val and XPG Asp1104His polymorphisms and digestive system cancer susceptibility, but the results were inconclusive. We performed a meta-analysis, using a comprehensive strategy based on the allele model and a model-free approach, to derive a more precise estimation of the relationship between XPC Ala499Val and XPG Asp1104His polymorphisms with digestive system cancer risk. For XPC Ala499Val, no significant cancer risk was found in the allele model (OR = 0.98, 95% CI: 0.86-1.11) and with model-free approach (ORG = 0.97, 95% CI: 0.83-1.13). For XPG Asp1104His, there was also no association between this polymorphism and cancer risk in the allele model (OR = 1.03, 95% CI: 0.96-1.11) and with the model-free approach (ORG = 1.04, 95% CI: 0.95-1.14). Therefore, this meta-analysis suggests that the XPC Ala499Val and XPG Asp1104His polymorphisms were not associated with digestive system cancer risk. Further large and well-designed studies are needed to confirm these findings.

  17. Infrared Structural Biology of Proteins: Development of Vibrational Structural Markers for Probing the Structural Dynamics of COO- of Asp/Glu in Proteins

    NASA Astrophysics Data System (ADS)

    Kang, Zhouyang; Xie, Aihua

    2013-03-01

    Asp and Glu often play critical roles in the active sites of proteins. Probing the structural dynamics of functionally important Asp and/or Glu provides crucial information for protein functionality. Time-resolved infrared structural biology offers strong advantages for its high structural sensitivity and broad dynamic range (ps to ks). In order to connect the vibrational frequencies to specific structures of COO- groups, such as the number, type, and geometry of hydrogen bond interactions, we develop two vibrational structural markers (VSM), built on the symmetric and asymmetric COO- stretching frequencies. Extensive quantum physics (density functional theory) based computational studies, combined with 13C isotopic editing of Asp/Glu and experimental FTIR data on Asp/Glu in proteins, are used to establish a unique correlation between the symmetric and asymmetric COO- vibrations with more than 10 types of hydrogen bonding interactions. Development of the COO- VSM markers enhances the power of time-resolved infrared structural biology for the study of functionally important structural dynamics of COO- in proteins, including rhodopsin for biological signaling, bacteriorhodopsin for proton transfer, photosystem II for energy transformation, and HIV protease for enzymatic catalysis.

  18. The Ising model in physics and statistical genetics.

    PubMed

    Majewski, J; Li, H; Ott, J

    2001-10-01

    Interdisciplinary communication is becoming a crucial component of the present scientific environment. Theoretical models developed in diverse disciplines often may be successfully employed in solving seemingly unrelated problems that can be reduced to similar mathematical formulation. The Ising model has been proposed in statistical physics as a simplified model for analysis of magnetic interactions and structures of ferromagnetic substances. Here, we present an application of the one-dimensional, linear Ising model to affected-sib-pair (ASP) analysis in genetics. By analyzing simulated genetics data, we show that the simplified Ising model with only nearest-neighbor interactions between genetic markers has statistical properties comparable to much more complex algorithms from genetics analysis, such as those implemented in the Allegro and Mapmaker-Sibs programs. We also adapt the model to include epistatic interactions and to demonstrate its usefulness in detecting modifier loci with weak individual genetic contributions. A reanalysis of data on type 1 diabetes detects several susceptibility loci not previously found by other methods of analysis.

  19. Conserved residues Asp16 and Pro24 of TnaC-tRNAPro participate in tryptophan induction of Tna operon expression.

    PubMed

    Cruz-Vera, Luis R; Yanofsky, Charles

    2008-07-01

    In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome. PMID:18424524

  20. [Affective dependency].

    PubMed

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy.

  1. [Affective dependency].

    PubMed

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy. PMID:23888587

  2. Validating Satellite Radar Altimetry Estimates of Antarctic sea ice Thickness Using the ASPeCt Data set

    NASA Astrophysics Data System (ADS)

    Giles, K. A.; Laxon, S. W.; Worby, T.

    2006-12-01

    Measurements of sea ice freeboard from spaceborne radar altimeters have been used to calculate Artic sea ice thickness on a basin wide scale during the winter. The same technique has the potential to be used in the Antarctic. The technique used to convert freeboard to thickness assumes hydrostatic equilibrium and uses estimates of snow depth and density and water and ice density from climatology. The nature of the Arctic climate means that the sea ice has a positive freeboard and that it becomes entirely snow free during the summer months, which simplifies the analysis of the radar return from the sea ice. However, in the Antarctic the situation may be more complicated with negative ice freeboards and flooded and refrozen snow resulting in inaccurate estimate of sea ice freeboard and therefore ice thickness. We present, for the first time, a comparison of estimates of Antarctic sea ice thickness calculated from satellite radar altimetry measurements of sea ice freeboard with ship observation of sea ice thickness from the ASPeCt data set. We describe the both the satellite and ship borne estimates of Antarctic sea ice thickness, the method used to compare the two data sets and outcome of the validation. We also assess the future potential of satellite radar altimetry to provide sea ice thickness in the Antarctic.

  3. The role of the Asn40Asp polymorphism of the mu opioid receptor gene (OPRM1) on alcoholism etiology and treatment: a critical review.

    PubMed

    Ray, Lara A; Barr, Christina S; Blendy, Julie A; Oslin, David; Goldman, David; Anton, Raymond F

    2012-03-01

    The endogenous opioid system has been implicated in the pathophysiology of alcoholism as it modulates the neurobehavioral effects of alcohol. A variant in the mu opioid receptor gene (OPRM1), the Asn40Asp polymorphism, has received attention as a functional variant that may influence a host of behavioral phenotypes for alcoholism as well as clinical response to opioid antagonists. This paper will review converging lines of evidence on the effect of the Asn40Asp SNP on alcoholism phenotypes, including: (i) genetic association studies; (ii) behavioral studies of alcoholism; (iii) neuroimaging studies; (iv) pharmacogenetic studies and clinical trials; and (v) preclinical animal studies. Together, these lines of research seek to elucidate the effects of this functional polymorphism on alcoholism etiology and treatment response.

  4. Molecular gallium arsenide phosphide clusters prepared from AsP(3), P(4), and [{GaC(SiMe(3))(3)}(4)].

    PubMed

    Cossairt, Brandi M; Cummins, Christopher C

    2010-11-01

    Treatment of AsP(3) with 0.75 equivalents of [{GaC(SiMe(3))(3)}(4)] resulted in selective insertion of three equivalents of {GaC(SiMe(3))(3)} into the three As--P bonds to give [As{GaC(SiMe(3))(3)}(3)P(3)] (1-As) with an intact cyclo-P(3) ring. This yellow compound has been characterized by NMR spectroscopy, combustion analysis, single-crystal X-ray diffraction, UV/Vis spectroscopy, Raman spectroscopy, and cyclic voltammetry (THF, 0.2 M [TBA][B(C(6)F(5))(4)]; TBA=tetrabutyl ammonium). Computational models of 1-As and the isomeric [P{GaC(SiMe(3))(3)}(3)AsP(2)] (1-P) have been investigated as well, revealing several interesting electronic features of these cage molecules. Following from the cyclic voltammetry studies of 1-As that highlight an irreversible two-electron reduction at -2.2 V versus Fc/Fc(+), treatment with one equivalent of [Mg(C(14)H(10))(thf)(3)] resulted in two-electron reduction to provide [As{GaC(SiMe(3))(3)}(3)P(3)Mg(thf)(3)] (2), in which the Mg(2+) ion has inserted into one of the P--P bonds of the cyclo-P(3) ring. It was also found that treatment of AsP(3) or P(4) with one equivalent of [{GaC(SiMe(3))(3)}(4)] resulted in formation of the quadruple insertion products [As{GaC(SiMe(3))(3)}(4)P(3)] (3) and [P{GaC(SiMe(3))(3)}(4)P(3)] (4), respectively.

  5. Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions*

    PubMed Central

    Lin, Wei; Wang, Ying; Han, Xiaobiao; Zhang, Zilong; Wang, Chengyuan; Wang, Jin; Yang, Huaiyu; Lu, Yinhua; Jiang, Weihong; Zhao, Guo-Ping; Zhang, Peng

    2014-01-01

    The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp50 residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg52 from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp50 with the highly conserved Arg52 and Thr9 in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation. PMID:24733389

  6. The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB.

    PubMed

    Hamilton, Christopher S; Spedaliere, Christopher J; Ginter, Joy M; Johnston, Murray V; Mueller, Eugene G

    2005-01-01

    All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.

  7. Nemaline myopathy-related skeletal muscle α-actin (ACTA1) mutation, Asp286Gly, prevents proper strong myosin binding and triggers muscle weakness.

    PubMed

    Ochala, Julien; Ravenscroft, Gianina; Laing, Nigel G; Nowak, Kristen J

    2012-01-01

    Many mutations in the skeletal muscle α-actin gene (ACTA1) lead to muscle weakness and nemaline myopathy. Despite increasing clinical and scientific interest, the molecular and cellular pathogenesis of weakness remains unclear. Therefore, in the present study, we aimed at unraveling these mechanisms using muscles from a transgenic mouse model of nemaline myopathy expressing the ACTA1 Asp286Gly mutation. We recorded and analyzed the mechanics of membrane-permeabilized single muscle fibers. We also performed molecular energy state computations in the presence or absence of Asp286Gly. Results demonstrated that during contraction, the Asp286Gly acts as a "poison-protein" and according to the computational analysis it modifies the actin-actin interface. This phenomenon is likely to prevent proper myosin cross-bridge binding, limiting the fraction of actomyosin interactions in the strong binding state. At the cell level, this decreases the force-generating capacity, and, overall, induces muscle weakness. To counterbalance such negative events, future potential therapeutic strategies may focus on the inappropriate actin-actin interface or myosin binding. PMID:23029319

  8. The P600-as-P3 hypothesis revisited: single-trial analyses reveal that the late EEG positivity following linguistically deviant material is reaction time aligned.

    PubMed

    Sassenhagen, Jona; Schlesewsky, Matthias; Bornkessel-Schlesewsky, Ina

    2014-10-01

    The P600, a late positive ERP component following linguistically deviant stimuli, is commonly seen as indexing structural, high-level processes, e.g. of linguistic (re)analysis. It has also been identified with the P3 (P600-as-P3 hypothesis), which is thought to reflect a systemic neuromodulator release facilitating behavioural shifts and is usually response time aligned. We investigated single-trial alignment of the P600 to response, a critical prediction of the P600-as-P3 hypothesis. Participants heard sentences containing morphosyntactic and semantic violations and responded via a button press. The elicited P600 was perfectly response aligned, while an N400 following semantic deviations was stimulus aligned. This is, to our knowledge, the first single-trial analysis of language processing data using within-sentence behavioural responses as temporal covariates. Results support the P600-as-P3 perspective and thus constitute a step towards a neurophysiological grounding of language-related ERPs.

  9. Flexible xxx-asp/asn and gly-xxx residues of equine cytochrome C in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

    PubMed

    Takayama, Mitsuo

    2012-01-01

    The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.

  10. An ecological vegetation-activated sludge process (V-ASP) for decentralized wastewater treatment: system development, treatment performance, and mathematical modeling.

    PubMed

    Yuan, Jiajia; Dong, Wenyi; Sun, Feiyun; Li, Pu; Zhao, Ke

    2016-05-01

    An environment-friendly decentralized wastewater treatment process that is comprised of activated sludge process (ASP) and wetland vegetation, named as vegetation-activated sludge process (V-ASP), was developed for decentralized wastewater treatment. The long-term experimental results evidenced that the vegetation sequencing batch reactor (V-SBR) process had consistently stable higher removal efficiencies of organic substances and nutrients from domestic wastewater compared with traditional sequencing batch reactor (SBR). The vegetation allocated into V-SBR system could not only remove nutrients through its vegetation transpiration ratio but also provide great surface area for microorganism activity enhancement. This high vegetation transpiration ratio enhanced nutrients removal effectiveness from wastewater mainly by flux enhancement, oxygen and substrate transportation acceleration, and vegetation respiration stimulation. A mathematical model based on ASM2d was successfully established by involving the specific function of vegetation to simulate system performance. The simulation results on the influence of operational parameters on V-ASP treatment effectiveness demonstrated that V-SBR had a high resistance to seasonal temperature fluctuations and influent loading shocking.

  11. The functional consequences of mis-sense mutations affecting an intra-molecular salt bridge in arylsulphatase A.

    PubMed Central

    Schestag, Frank; Yaghootfam, Afshin; Habetha, Matthias; Poeppel, Peter; Dietz, Frank; Klein, Roger A; Zlotogora, Joel; Gieselmann, Volkmar

    2002-01-01

    Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. We describe the functional consequences of three mis-sense mutations in the arylsulphatase A gene (Asp-335-Val, Arg-370-Trp and Arg-370-Gln), affecting an apparent intramolecular Asp-335 to Arg-370 salt bridge, and interpret the effects and clinical consequences on the basis of the three-dimensional structure of arylsulphatase A. Asp-335-Val and Arg-370-Trp substitutions each cause a complete loss of enzyme activity and are associated with the most severe form of the human disease, whereas the Arg-370-Gln-substituted enzyme retains some residual activity, being found in a patient suffering from the milder juvenile form of the disease. Detailed analysis reveals that formation of the apparent salt bridge depends critically on the presence of aspartic acid and arginine residues at positions 335 and 370, respectively. Substitution by various other amino acids, including glutamic acid and lysine, affects enzyme function severely. Biosynthesis and immunoprecipitation studies indicate that the Asp-335-Val substitution affects folding of arylsulphatase A more severely than either the Arg-370-Trp or Arg-370-Gln substitutions. In vitro mutagenesis data show that clinical severity correlates with the space occupied by residue 370. The combination with structural data suggests that the bulky tryptophan residue broadens the cleft held together by the apparent salt bridge, whereas the smaller glutamine residue still allows the cleft to close, yielding a less severely affected enzyme. The position of residue 370 in the three-dimensional structure of the enzyme provides a plausible explanation for the differing severities in loss of enzyme function caused by the mutations and thus the clinical phenotype. PMID:12086582

  12. Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model.

    PubMed

    Sawyer, R T; Garner, R E; Hudson, J A

    1992-01-01

    The isolated perfused mouse liver model was used to study the effect of Arg-Gly-Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD-treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells.

  13. An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

    NASA Technical Reports Server (NTRS)

    Yamakawa, K.; Duncan, R.; Hruska, K. A.

    1994-01-01

    We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

  14. Molecular basis of the clotting defect in a bleeding patient missing the Asp-185 codon in the factor X gene.

    PubMed

    Lu, Qiuya; Yang, Likui; Manithody, Chandrashekhara; Wang, Xuefeng; Rezaie, Alireza R

    2014-11-01

    Factor X (FX) is a vitamin K-dependent plasma zymogen, which following activation to factor Xa (FXa), converts prothrombin to thrombin in the blood clotting cascade. It was recently demonstrated that a natural variant of FX carrying the Asp-185 deletion (FX-D185del, chymotrypsinogen numbering) was associated with mild bleeding in a patient with severe FX deficiency. In this study, we expressed FX-D185del in mammalian cells and characterized its properties in appropriate kinetic assays in purified systems. We discovered that while the FX variant can be normally activated by physiological activators; both amidolytic and proteolytic activities of the mutant are dramatically impaired. Interestingly, factor Va (FVa) significantly improved the proteolytic defect when the mutant protease was assembled into the prothrombinase complex. Thus, in contrast to >50-fold catalytic defect in the absence of FVa, the variant activated prothrombin with only ~2.5-fold decreased catalytic efficiency in the presence of the cofactor. The FXa variant dramatically lost its susceptibility to inhibition by antithrombin and tissue factor pathway inhibitor, thus exhibiting ~2-3 orders of magnitude lower reactivity with the plasma inhibitors. Further studies revealed that Na(+) no longer activates the variant protease, suggesting that the functionally important allosteric linkage between the Na(+)-binding and the P1-binding sites of the protease has been eliminated. These results suggest that the lower catalytic efficiency of FXa-D185del in the bleeding patient may be partially compensated by the loss of its reactivity with plasma inhibitors, possibly explaining the basis for the paradoxical severe FX deficiency with only mild bleeding tendency for this mutation. PMID:25179519

  15. Targeting effect of PEGylated liposomes modified with the Arg-Gly-Asp sequence on gastric cancer.

    PubMed

    Ding, Jie; Feng, Min; Wang, Feng; Wang, Hao; Guan, Wenxian

    2015-10-01

    Previous studies have demonstrated that the α5β1 integrin-mediated interaction with fibronectin (FN) occurs through the Arg-Gly-Asp (RGD) cell-binding sequence in repeat III10. Indocyanine green (ICG) is a near-infrared (NIR) optical dye that has been approved by the US Food and Drug Administration. In the present study, we developed an RGD-modified PEGylated liposome-encapsulated ICG (RGD-PLS-ICG) system mediated by integrin. RGD was conjugated covalently to the distal end of DSPE-PEG2000-NH2 lipid by amide binding. The characteristics and stability of the prepared liposomes were assessed. In vitro, SGC7901 cells with high expression of integrin α5β1 were selected by polymerase chain reaction (PCR) and western blotting. To confirm the targeting efficacies to gastric cancer, coumarin-6 was encapsulated as a fluorescent probe for in vitro study, and the targeting effect of RGD was detected by flow cytometry and confocal microscopy. In vivo, the bio distribution of RGD-PLS-ICG was studied by an in vivo imaging system in the tumor model. RGD-PLS-ICG and PLS-ICG had a higher UV absorbance spectrum and stability than free-ICG. Confocal microscopy and flow cytometry demonstrated that RGD-PLS-encapsulated coumarin-6 was efficiently associated with the SGC7901 cells, while limited interaction was found for the other groups. Moreover, the in vivo imaging of the liposomes indicated that RGD-PLS-ICG achieved more accumulation in the tumor tissues when compared with PLS-ICG. The significant in vitro and in vivo results suggest that RGD-PLS-ICG may be a promising fluorescent dye delivery system for targeting gastric cancer cell overexpression of integrin. PMID:26238930

  16. Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

    PubMed Central

    Bonnet, R.; Dutour, C.; Sampaio, J. L. M.; Chanal, C.; Sirot, D.; Labia, R.; De Champs, C.; Sirot, J.

    2001-01-01

    Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two blaCTX-M genes encoding β-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the blaCTX-M-9 gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated blaCTX-M-16, observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240→Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 μg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 μg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher kcat for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two blaCTX-M-9 genes and the blaCTX-M-16 gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M β-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum β-lactamases in Brazil. PMID:11451684

  17. Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction

    PubMed Central

    McDonald, Denise M.; Coleman, Gary; Bhatwadekar, Ashay; Gardiner, Tom A.

    2009-01-01

    Purpose Advanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endothelial cells (RMECs) growing on AGE-modified fibronectin (FN) and determined how this has an impact on cell-substrate interactions and downstream oxidative responses and cell survival. Methods RMECs were grown on methylglyoxal-modified FN (AGE-FN) or native FN as a control. RMEC attachment and spreading was quantified. In a separate treatment, the AGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide added before seeding. Phosphorylation of focal adhesion kinase (FAK) and α5β1 integrin localization was assessed and apoptosis evaluated. In a subset of RMECs that remained attached to the AGE-FN substrate, the production of superoxide (O2-) was assayed using dihydroethidium (DHE) fluorescence or lucigenin, in the presence or absence of NADPH. The specificity of the O2- assays was confirmed by inhibition in the presence of polyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediated changes to mRNAs encoding key basement membrane proteins and regulatory enzymes were investigated using real-time RT–PCR. Results AGE-FN reduced RMEC attachment and spreading when compared to FN controls (p<0.001). RGDS peptide enhanced cell attachment on AGE-FN (p<0.001), while the scrambled peptide had no effect. FAK phosphorylation in AGE-exposed RMECs was reduced in a time-dependent fashion, while α5β1 integrin-immunoreactivity became focal at the basal membrane. AGE-exposure induced apoptosis, a response significantly prevented by RGDS peptide. AGE-exposure caused a significant increase in basal O2- and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN also increased basement membrane associated mRNA expression (p<0.05). Conclusions AGE substrate modifications

  18. Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase.

    PubMed

    Le Roy, Katrien; Lammens, Willem; Verhaest, Maureen; De Coninck, Barbara; Rabijns, Anja; Van Laere, André; Van den Ende, Wim

    2007-11-01

    Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

  19. Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase1[C

    PubMed Central

    Le Roy, Katrien; Lammens, Willem; Verhaest, Maureen; De Coninck, Barbara; Rabijns, Anja; Van Laere, André; Van den Ende, Wim

    2007-01-01

    Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs. PMID:17873089

  20. Identification of a Novel Mutation in the CYBB Gene, p.Asp378Gly, in a Patient With X-linked Chronic Granulomatous Disease.

    PubMed

    Song, Sang-Mi; Park, Mi-Ran; Kim, Do-Soo; Kim, Jihyun; Kim, Yae-Jean; Ki, Chang-Seok; Ahn, Kangmo

    2014-07-01

    Chronic granulomatous disease (CGD) is a rare immunodeficiency disease, which is characterized by the lack of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. The disease presents leukocytosis, anemia, hypergammaglobulinemia, and granuloma formation of the skin, lung, or lymph nodes. The mutation of the CYBB gene encoding gp91phox, located on chromosome Xp21.1 is one of the causes of CGD. We report a patient with X-linked CGD who carried a novel mutation, a c.1133A>G (paAsp378Gly) missense mutation, in the CYBB gene. PMID:24991462

  1. Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions.

    PubMed

    Avaeva, S M; Velichko, T I; Vorobyeva, N N; Kurilova, S A; Nazarova, T I; Sklyankina, V A

    1999-02-01

    Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration. PMID:10187907

  2. Transesterification and amide cis-trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc-Asp(Dpa)-OBzl.

    PubMed

    Niklas, Nicole; Zahl, Achim; Alsfasser, Ralf

    2007-01-01

    The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions. PMID:17160185

  3. Capillary electrophoresis analysis of the degradation of the aspartyl tripeptide Phe-Asp-GlyOH at pH 2.0 and 7.4 under forced conditions.

    PubMed

    Conrad, Uwe; Taichrib, Angelina; Neusüss, Christian; Scriba, Gerhard K E

    2010-02-01

    The degradation of the tripeptide l-Phe-alpha-l-Asp-GlyOH was studied at 80 degrees C and pH 2.0 and 7.4 by capillary electrophoresis. Separation of most known as well as unknown degradation products was achieved in a 50mM sodium phosphate buffer, pH 3.0. The diastereomers l-Phe-alpha-l-Asp-GlyOH/l-Phe-alpha-d-Asp-GlyOH could only be separated upon addition of 16mg/ml carboxymethyl-beta-cyclodextrin and 5% acetonitrile to the background electrolyte. Compound identification was performed by capillary electrophoresis-electrospray ionization-mass spectrometry. In addition to Asp isomerization and epimerization products as well as hydrolysis products four diketopiperazine derivatives were identified. Moreover, two degradation products were observed containing the amino acids Asp, Gly and Phe but the unequivocal assignment could not be accomplished based on the mass spectra. Following validation with regard to linearity, range, limit of detection, limit of quantitation and precision the assay was applied to the analysis of the incubation solutions. While peptide backbone hydrolysis dominated at pH 2.0, isomerization and enantiomerization yielding beta-Asp and d-Asp peptides as well as cyclization to diketopiperazine derivatives were observed at pH 7.4. The diketopiperazines were the dominant reaction products amounting to about 85% of the compounds detected after the maximal incubation time of 240h. A kinetic model was used to fit the concentration versus time data.

  4. Gly322Asp and Asn127Ser single nucleotide polymorphisms (SNPs) of hMSH2 mismatch repair gene and the risk of triple-negative breast cancer in Polish women.

    PubMed

    Smolarz, Beata; Makowska, Marianna; Samulak, Dariusz; Michalska, Magdalena M; Romanowicz, Hanna

    2015-03-01

    Triple-negative breast cancer (TNBC) is characterised by worse clinical outcome and poor prognosis. The alterations in the oncogenes and tumor suppressor genes as well as microsatellite instability (MSI) have been associated with breast cancer development. It is knowledge that the most common mechanism inducing MSI in many cancer is genomic rearrangements found in the hMSH2 (human MutS homolog 2) gene. In this report we genotyped two polymorphisms of hMSH2 DNA repair gene in 70 TNBC patients and 70 age-matched cancer-free women using RFLP-PCR. The following polymorphisms were studied: an A/G transition at 127 positions producing an Asn/Ser substitution at codon 127 (the Asn127Ser polymorphism, rs17217772) and a G/A transition at 1032 position resulting in a Gly/Asp change at codon 322 (the Gly322Asp polymorphism, rs4987188). We found an association between the hMSH2 Asp/Asp and Gly/Asp genotypes and TNBC occurence. Variant Asp allele of hMSH2 decreased cancer risk [odds ratio (OR) 0.11; 95 % confidence interval (CI) 0.05-0.21]. The risk of TNBC in the carriers of the Gly322Gly-Asn127Ser combined genotype was increased (OR 3.71; 95 % CI 1.36-10.10). However the risk of TNBC was not alter by polymorphism Asn127Ser of the hMSH2 gene. The Gly322Asp polymorphism of the hMSH2 gene may be linked with TNBC occurrence in Polish women.

  5. Multimodal MRI and (31)P-MRS investigations of the ACTA1(Asp286Gly) mouse model of nemaline myopathy provide evidence of impaired in vivo muscle function, altered muscle structure and disturbed energy metabolism.

    PubMed

    Gineste, Charlotte; Duhamel, Guillaume; Le Fur, Yann; Vilmen, Christophe; Cozzone, Patrick J; Nowak, Kristen J; Bendahan, David; Gondin, Julien

    2013-01-01

    Nemaline myopathy (NM), the most common non-dystrophic congenital disease of skeletal muscle, can be caused by mutations in the skeletal muscle α-actin gene (ACTA1) (~25% of all NM cases and up to 50% of severe forms of NM). Muscle function of the recently generated transgenic mouse model carrying the human Asp286Gly mutation in the ACTA1 gene (Tg(ACTA1)(Asp286Gly)) has been mainly investigated in vitro. Therefore, we aimed at providing a comprehensive picture of the in vivo hindlimb muscle function of Tg(ACTA1)(Asp286Gly) mice by combining strictly noninvasive investigations. Skeletal muscle anatomy (hindlimb muscles, intramuscular fat volumes) and microstructure were studied using multimodal magnetic resonance imaging (Dixon, T2, Diffusion Tensor Imaging [DTI]). Energy metabolism was studied using 31-phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (1-150 Hz) and a fatigue protocol (6 min-1.7 Hz). Tg(ACTA1)(Asp286Gly) mice showed a mild muscle weakness as illustrated by the reduction of both absolute (30%) and specific (15%) maximal force production. Dixon MRI did not show discernable fatty infiltration in Tg(ACTA1)(Asp286Gly) mice indicating that this mouse model does not reproduce human MRI findings. Increased T2 values were observed in Tg(ACTA1)(Asp286Gly) mice and might reflect the occurrence of muscle degeneration/regeneration process. Interestingly, T2 values were linearly related to muscle weakness. DTI experiments indicated lower λ2 and λ3 values in Tg(ACTA1)(Asp286Gly) mice, which might be associated to muscle atrophy and/or the presence of histological anomalies. Finally (31)P-MRS investigations illustrated an increased anaerobic energy cost of contraction in Tg(ACTA1)(Asp286Gly) mice, which might be ascribed to contractile and non-contractile processes. Overall, we provide a unique set of information about the anatomic, metabolic and functional

  6. Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen.

    PubMed

    Di Nardo, Giovanna; Dell'Angelo, Valentina; Catucci, Gianluca; Sadeghi, Sheila J; Gilardi, Gianfranco

    2016-07-15

    This paper reports the structure of the double mutant Asp251Gly/Gln307His (named A2) generated by random mutagenesis, able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. The 3D structure of the substrate-free mutant shows a conformation similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on I-helix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules. The impact of these structural features on thermal stability, reduction potential and electron transfer is investigated. The data demonstrate that a single mutation far from the active site triggers an increase in protein flexibility in a key region, shifting the conformational equilibrium toward the closed form that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted. PMID:26718083

  7. Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

    PubMed

    Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

    2016-06-01

    Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. PMID:27276251

  8. A QM/MM study of Kemptide phosphorylation catalyzed by protein kinase A. The role of Asp166 as a general acid/base catalyst.

    PubMed

    Pérez-Gallegos, Ayax; Garcia-Viloca, Mireia; González-Lafont, Àngels; Lluch, José M

    2015-02-01

    In this work a theoretical study of the γ-phosphoryl group transfer from ATP to Ser17 of the synthetic substrate Kemptide (LRRASLG) in protein kinase A (PKA) has been carried out with a solvated model of the PKA-Mg2ATP-Kemptide system based on the X-ray crystallographic structure. We have used high levels (B3LYP/MM and MP2/MM) of theory to determine the overall reaction paths of the so-called concerted loose mechanism trying to clarify some aspects of that mechanism still under debate. Our calculations demonstrate for the first time in a complete model of the ternary system the viability of the final step of the catalytic mechanism in which the protonation of the phosphokemptide product by Asp166 takes place. Asp166 is a base catalyst that abstracts the HγSer17 of Kemptide thus facilitating the phosphoryl transfer, but it also acts as an acid catalyst by donating the proton just accepted from Ser17 to the O2γATP atom of the phosphoryl group.

  9. Simultaneous determination of a novel oral Janus kinase inhibitor ASP015K and its sulfated metabolite in rat plasma using LC-MS/MS.

    PubMed

    Oda, Kazuo; Mera, Katsumi; Nagasaka, Yasuhisa; Tokoro, Kazumi

    2015-07-01

    A sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed for determining the concentrations of novel Janus kinase inhibitor ASP015K and its sulfated metabolite M2 in rat plasma. This method involves solid-phase extraction (SPE) from 25 μL of rat plasma. LC separation was performed on an Inertsil PH-3 column (100 mm L ×4.6 mm I.D., 5 µm) with a mobile phase consisting of 10 mM ammonium acetate and methanol under linear gradient conditions. Analytes were introduced to the LC-MS/MS through an electrospray ionization source and detected in positive-ion mode using selected reaction monitoring. Standard curves were linear from 0.25 to 500 ng/mL (r ≥0.9964). This assay enabled quantification of ASP015K and M2 at a concentration as low as 0.25 ng/mL in rat plasma. Validation data demonstrated that the method is selective, sensitive and accurate. Further, we also successfully applied this method to a preclinical pharmacokinetic study in rats.

  10. Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

    PubMed

    Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

    2016-06-01

    Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

  11. Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution.

    PubMed

    Fu, Zhirong; Thorpe, Michael; Akula, Srinivas; Hellman, Lars

    2016-01-01

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus. PMID:27152961

  12. Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution

    PubMed Central

    Fu, Zhirong; Thorpe, Michael; Akula, Srinivas; Hellman, Lars

    2016-01-01

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus. PMID:27152961

  13. Computational Insights into Substrate and Site Specificities, Catalytic Mechanism, and Protonation States of the Catalytic Asp Dyad of β-Secretase

    PubMed Central

    Barman, Arghya; Prabhakar, Rajeev

    2014-01-01

    In this review, information regarding substrate and site specificities, catalytic mechanism, and protonation states of the catalytic Asp dyad of β-secretase (BACE1) derived from computational studies has been discussed. BACE1 catalyzes the rate-limiting step in the generation of Alzheimer amyloid beta peptide through the proteolytic cleavage of the amyloid precursor protein. Due to its biological functioning, this enzyme has been considered as one of the most important targets for finding the cure for Alzheimer's disease. Molecular dynamics (MD) simulations suggested that structural differences in the key regions (inserts A, D, and F and the 10s loop) of the enzyme are responsible for the observed difference in its activities towards the WT- and SW-substrates. The modifications in the flap, third strand, and insert F regions were found to be involved in the alteration in the site specificity of the glycosylphosphatidylinositol bound form of BACE1. Our QM and QM/MM calculations suggested that BACE1 hydrolyzed the SW-substrate more efficiently than the WT-substrate and that cleavage of the peptide bond occurred in the rate-determining step. The results from molecular docking studies showed that the information concerning a single protonation state of the Asp dyad is not enough to run an in silico screening campaign. PMID:25309776

  14. Electrostatics of the photosynthetic bacterial reaction center. Protonation of Glu L 212 and Asp L 213 - A new method of calculation.

    PubMed

    Ptushenko, Vasily V; Cherepanov, Dmitry A; Krishtalik, Lev I

    2015-12-01

    Continuum electrostatic calculation of the transfer energies of anions from water into aprotic solvents gives the figures erroneous by order of magnitude. This is due to the hydrogen bond disruption that suggests the necessity to reconsider the traditional approach of the purely electrostatic calculation of the transfer energy from water into protein. In this paper, the method combining the experimental estimates of the transfer energies from water into aprotic solvent and the electrostatic calculation of the transfer energies from aprotic solvent into protein is proposed. Hydrogen bonds between aprotic solvent and solute are taken into account by introducing an imaginary aprotic medium incapable to form hydrogen bonds with the solute. Besides, a new treatment of the heterogeneous intraprotein dielectric permittivity based on the microscopic protein structure and electrometric measurements is elaborated. The method accounts semi-quantitatively for the electrostatic effect of diverse charged amino acid substitutions in the donor and acceptor parts of the photosynthetic bacterial reaction center from Rhodobacter sphaeroides. Analysis of the volatile secondary acceptor site QB revealed that in the conformation with a minimal distance between quinone QB and Glu L 212 the proton uptake upon the reduction of QB is prompted by Glu L 212 in alkaline and by Asp L 213 in slightly acidic regions. This agrees with the pH dependences of protonation degrees and the proton uptake. The method of pK calculation was applied successfully also for dissociation of Asp 26 in bacterial thioredoxin. PMID:26210154

  15. Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis.

    PubMed

    Zhang, Yu; Yu, Haixia; Wu, Yujiao; Zhao, Wenyan; Yang, Min; Jing, Huanwang; Chen, Anjia

    2014-10-01

    In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-β-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HP-β-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE.

  16. Appearance of Planktothrix rubescens Bloom with [D-Asp3, Mdha7]MC–RR in Gravel Pit Pond of a Shallow Lake-Dominated Area

    PubMed Central

    Vasas, Gábor; Farkas, Oszkár; Borics, Gábor; Felföldi, Tamás; Sramkó, Gábor; Batta, Gyula; Bácsi, István; Gonda, Sándor

    2013-01-01

    Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing genera in temperate lakes. The reddish color of cyanobacterial blooms viewed in a gravel pit pond with the appearance of a dense 3 cm thick layer (biovolume: 28.4 mm3 L−1) was an unexpected observation in the shallow lake-dominated alluvial region of the Carpathian Basin. [d-Asp3, Mdha7]MC–RR was identified from the blooms sample by MALDI-TOF and NMR. Concentrations of [d-Asp3, Mdha7]MC–RR were measured by capillary electrophoresis to compare the microcystin content of the field samples and the isolated, laboratory-maintained P. rubescens strain. In analyzing the MC gene cluster of the isolated P. rubescens strain, a deletion in the spacer region between mcyE and mcyG and an insertion were located in the spacer region between mcyT and mcyD. The insertion elements were sequenced and partly identified. Although some invasive tropical cyanobacterial species have been given a great deal of attention in many recent studies, our results draw attention to the spread of the alpine organism P. rubescens as a MC-producing, bloom-forming species. PMID:24351711

  17. Evidence for linkage of bipolar disorder to chromosome 18 with a parent-of-origin effect

    SciTech Connect

    Stine, O.C.; Xu, Jianfeng; McMahon, F.J.

    1995-12-01

    A susceptibility gene on chromosome18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sib-pair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = .0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father`s sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; {theta} = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study. 49 refs., 2 figs., 5 tabs.

  18. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

    PubMed

    Luo, Feijun; Brooks, David G; Ye, Hongtao; Hamoudi, Rifat; Poulogiannis, George; Patek, Charles E; Winton, Douglas J; Arends, Mark J

    2009-10-01

    Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant

  19. Molecular and biochemical characterization of CTX-M-131, a natural Asp240Gly variant derived from CTX-M-2, produced by a Providencia rettgeri clinical strain in São Paulo, Brazil.

    PubMed

    Dropa, Milena; Ghiglione, Barbara; Matté, Maria Helena; Balsalobre, Livia Carminato; Lincopan, Nilton; Matté, Glavur Rogério; Gutkind, Gabriel; Power, Pablo

    2015-03-01

    CTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in a Providencia rettgeri clinical strain from Brazil. Molecular analysis showed that blaCTX-M-131 was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime.

  20. Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate.

    PubMed

    Gat, Y; Friedman, N; Sheves, M; Ottolenghi, M

    1997-04-01

    Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the

  1. A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

    PubMed

    Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

    2013-07-12

    Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG. PMID:23733182

  2. Effect of changing the position and orientation of Asp85 relative to the protonated Schiff base within the retinal cavity on the rate of photoisomerization in bacteriorhodopsin

    SciTech Connect

    Song, L.; El-Sayed, M.A.; Lanyi, J.K.

    1996-06-13

    Replacement of either Val49 with Ala or Ala53 with Val by site-specific mutagenesis is known to change the position and orientation of the protonated Schiff base relative to Asp85 within the retinal cavity of bacteriorhodopsin (bR). The effect of mutation on the rate of the subpicosecond photoisomerization of retinal in bR is examined by using a pump-probe technique. A decrease in the rate of photoisomerization of retinal in V49A and A53V is observed. This is discussed in terms of the previously proposed mechanism of the protein catalysis of retinal photoisomerization process in bR. 17 refs., 2 figs., 1 tab.

  3. Visualization of implanted GL261 glioma cells in living mouse brain slices using fluorescent 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+)

    PubMed Central

    Kucheryavykh, Lilia Y.; Kucheryavykh, Yuriy V.; Rolón-Reyes, Kimberleve; Skatchkov, Serguei N.; Eaton, Misty J.; Cubano, Luis A.; Inyushin, Mikhail

    2013-01-01

    Here we describe a new method of glioma cell visualization in living brain slices that can be used for evaluation of tumor size or visualization of internal tumor structures. Glial cells, as well as glioma cells of glial origin, express high levels of organic cation transporters. We demonstrate that application of a fluorescent substrate for these transporters 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+) to the incubation medium leads to quick accumulation of fluorescence in glioma cells during early developmental stages and in astrocytes, but not in neurons. Stained brain slices can be immediately investigated using confocal or fluorescence microscopy. Glioma and glial cells can be discriminated from each other due to their different morphology. The method described has an advantage of staining living tissue and is simple to perform. PMID:23570046

  4. Water Dynamics in Egg White Peptide, Asp-His-Thr-Lys-Glu, Powder Monitored by Dynamic Vapor Sorption and LF-NMR.

    PubMed

    Yang, Shuailing; Liu, Xuye; Jin, Yan; Li, Xingfang; Chen, Feng; Zhang, Mingdi; Lin, Songyi

    2016-03-16

    Water absorbed into the bulk amorphous structure of peptides can have profound effects on their properties. Here, we elucidated water dynamics in Asp-His-Thr-Lys-Glu (DHTKE), an antioxidant peptide derived from egg white ovalbumin, using water dynamic vapor sorption (DVS) and low-field nuclear magnetic resonance (LF-NMR). The DVS results indicated that parallel exponential kinetics model fitted well to the data of sorption kinetics behavior of DHTKE. Four different proton fractions with different mobilities were identified based on the degree of interaction between peptide and water. The water could significantly change the proton distribution and structure of the sample. The different phases of moisture absorption were reflected in the T2 parameters. In addition, the combined water content was dominant in the hygroscopicity of DHTKE. This study provides an effective real-time monitoring method for water mobility and distribution in synthetic peptides, and this method may have applications in promoting peptide quality assurance. PMID:26915514

  5. Effects of Zn2+ Binding on the Structural and Dynamic Properties of Amyloid Β Peptide Associated with Alzheimer’s Disease: Asp1 or Glu11?

    PubMed Central

    2013-01-01

    Extensive experimental and computational studies have suggested that multiple Zn2+ binding modes in amyloid β (Aβ) peptides could exist simultaneously. However, consistent results have not been obtained for the effects of Zn2+ binding on Aβ structure, dynamics, and kinetics in particular. Some key questions such as why it is so difficult to distinguish the polymorphic states of metal ions binding to Aβ and what the underlying rationale is, necessitate elucidation. In this work, two 3N1O Zn2+ binding modes were constructed with three histidines (His6, His13, and His14), and Asp1/Glu11 of Aβ40 coordinated to Zn2+. Results from molecular dynamics simulations reveal that the conformational ensembles of different Zn2+-Aβ40 complexes are nonoverlapping. The formation of turn structure and, especially, the salt bridge between Glu22/Asp23 and Lys28 is dependent on specific Zn2+ binding mode. Agreement with available NMR observations of secondary and tertiary structures could be better achieved if the two simulation results are considered together. The free energy landscape constructed by combining both conformations of Aβ40 indicates that transitions between distinct Aβ40 conformations thar are ready for Zn2+ binding could be possible in aqueous solution. Markov state model analyses reveal the complex network of conformational space of Aβ40 modeulated by Zn2+ binding, suggesting various misfolding pathways. The binding free energies evaluated using a combination of quantum mechanics calculations and the MM/3D-RISM method suggest that Glu11 is the preferred oxygen ligand of Zn2+. However, such preference is dependent on the relative populations of different conformations with specific Zn2+ binding modes, and therefore could be shifted when experimental or simulation conditions are altered. PMID:23947440

  6. Accurate spectroscopic calculations of the 17 Λ-S and 59 Ω states of the AsP molecule including the spin-orbit coupling effect

    NASA Astrophysics Data System (ADS)

    Shi, Deheng; Liu, Qionglan; Wang, Shuai; Sun, Jinfeng; Zhu, Zunlue

    2015-01-01

    The potential energy curves (PECs) of 59 Ω states generated from the 17 Λ-S states (X1Σ+, a3Σ+, 15Σ+, b3Δ, c3Π, 15Π, 25Σ+, 23Δ, 23Π, 33Σ+, A1Π, 23Σ+, 35Σ+, 17Σ+, 15Δ, 25Δ, and 25Π) of AsP molecule are studied for the first time for internuclear separations from about 0.10 to 1.10 nm. All the Λ-S states are contributed to the first three dissociation channels of AsP molecule except for the A1Π. The 23Σ+, 35Σ+, 17Σ+, 15Δ, 25Δ, and 25Π are found to be the repulsive states. The a3Σ+, 15Π, b3Δ, 17Σ+, 15Δ, 25Δ, and 25Π are found to be the inverted states. Each of the 33Σ+, c3Π, 23Π, 15Π, and 15Σ+ states has one potential barrier. The PECs are calculated by the CASSCF method, which is followed by the internally contracted MRCI approach with Davidson correction. Core-valence correlation and scalar relativistic corrections are included. The convergent behavior of present calculations is discussed with respect to the basis set and level of theory. The spin-orbit coupling effect is accounted for. All these PECs are extrapolated to the complete basis set limit. The spectroscopic parameters are evaluated for the bound states involved, and are compared with available measurements. Excellent agreement has been found between the present results and the measurements. It shows that the spectroscopic parameters reported in this paper can be expected to be reliably predicted ones. The conclusion is gained that the effect of spin-orbit coupling on the spectroscopic parameters is not obvious for all the Λ-S bound states except for few ones such as 15Σ+ and c3Π.

  7. Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not

    PubMed Central

    Rafique, Nazmi; Goodman, Michael Christopher; Idborg, Helena; Bergqvist, Filip; Jakobsson, Per-Johan

    2016-01-01

    Introduction Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism. Aim The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1. Materials and Methods Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling. Results and Conclusions Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity. PMID:27684486

  8. Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

    PubMed Central

    Cecilio, Alzira B.; Caldas, Sergio; De Oliveira, Raiana A.; Santos, Arthur S. B.; Richardson, Michael; Naumann, Gustavo B.; Schneider, Francisco S.; Alvarenga, Valeria G.; Estevão-Costa, Maria I.; Fuly, Andre L.; Eble, Johannes A.; Sanchez, Eladio F.

    2013-01-01

    We report the detailed molecular characterization of two PLA2s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA2s, named BlK-PLA2 and BlD-PLA2, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA2s, and show a high degree of sequence similarity to homologous snake venom PLA2s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA2s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA2s were assessed by quantitative Real-Time PCR. Bl-PLA2s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA2s as tools of research. PMID:24131891

  9. Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 induces penile erection via brain and spinal melanocortin receptors.

    PubMed

    Wessells, H; Hruby, V J; Hackett, J; Han, G; Balse-Srinivasan, P; Vanderah, T W

    2003-01-01

    Penile erection induced by alpha-melanocyte-stimulating hormone and melanocortin receptors (MC-R) in areas of the spinal cord and periphery has not been demonstrated. To elucidate sites of the proerectile action of melanocortin peptides, in awake male rats we administered the MC-R agonist Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2) (MT-II) i.c.v., intrathecal (i.th.) and i.v. and scored penile erection and yawning. Injection of the MC-R antagonist Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2) (SHU-9119) i.c.v. or i.th. in combination with i.th. MT-II differentiated spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracavernous pressure responses to intracavernosal injection of MT-II in the anesthetized rat.I.c.v., i.th., and i.v. MT-II induced penile erections in a dose-dependent fashion. Yawning was observed with i.c.v. and i.v. MT-II, while spinal injection did not produce this behavior. Intrathecal delivery of MT-II to the lumbosacral spinal cord was more efficacious in inducing erections than i.c.v. or i.v. administration; SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracavernous pressure nor augmented neurostimulated erectile responses. We confirmed the central proerectile activity of MT-II and demonstrated that in addition to a site of action in the brain, the distal spinal cord contains melanocortin receptors that can initiate penile erection independent of higher centers. These results provide new insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction.

  10. Model-free linkage analysis of a binary trait.

    PubMed

    Xu, Wei; Bull, Shelley B; Mirea, Lucia; Greenwood, Celia M T

    2012-01-01

    Genetic linkage analysis aims to detect chromosomal regions containing genes that influence risk of specific inherited diseases. The presence of linkage is indicated when a disease or trait cosegregates through the families with genetic markers at a particular region of the genome. Two main types of genetic linkage analysis are in common use, namely model-based linkage analysis and model-free linkage analysis. In this chapter, we focus solely on the latter type and specifically on binary traits or phenotypes, such as the presence or absence of a specific disease. Model-free linkage analysis is based on allele-sharing, where patterns of genetic similarity among affected relatives are compared to chance expectations. Because the model-free methods do not require the specification of the inheritance parameters of a genetic model, they are preferred by many researchers at early stages in the study of a complex disease. We introduce the history of model-free linkage analysis in Subheading 1. Table 1 describes a standard model-free linkage analysis workflow. We describe three popular model-free linkage analysis methods, the nonparametric linkage (NPL) statistic, the affected sib-pair (ASP) likelihood ratio test, and a likelihood approach for pedigrees. The theory behind each linkage test is described in this section, together with a simple example of the relevant calculations. Table 4 provides a summary of popular genetic analysis software packages that implement model-free linkage models. In Subheading 2, we work through the methods on a rich example providing sample software code and output. Subheading 3 contains notes with additional details on various topics that may need further consideration during analysis.

  11. Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

    NASA Astrophysics Data System (ADS)

    Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

    2014-05-01

    Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

  12. Dietary fatty acid composition affects aminopeptidase activities in the testes of mice.

    PubMed

    Arechaga, Garbiñe; Prieto, Isabel; Segarra, Ana B; Alba, Francisco; Ruiz-Larrea, María B; Ruiz-Sanz, José I; de Gasparo, Marc; Ramirez, Manuel

    2002-04-01

    The autocrine/paracrine control mechanisms of local factors, such as the renin-angiotensin system and the thyrotropin-releasing hormone (TRH), seem to play a relevant role in testicular physiology. It has been proposed that dietary fat composition influences male reproductive function modifying the cholesterol-phospholipid composition of testicular plasma membranes. Modifications in the composition and physical properties of the membranes may lead to alterations in the activities of membrane-bound (M-B) enzymes. We have previously demonstrated that cholesterol and steroid hormones affect aminopeptidase (AP) activities. Dietary fatty acids with different degrees of saturation modified AP activities in the serum of mice and an olive oil supplemented diet influenced the AP activities in the testes of mice. We hypothesized that the modification of dietary fat composition may affect angiotensin- [glutamyl-AP (GluAP), aspartyl-AP (AspAP)] and TRH- [pyroglutamyl-AP (pGluAP)] degrading activities in the testis. In this study, we investigated the effect of diets supplemented with sunflower oil (SFO), fish oil (FO), olive oil (OO), lard (L) or coconut oil (CO) on soluble (Sol) and M-B GluAP, AspAP and pGluAP in mice testis, using arylamides as substrates. Sol GluAP activity did not show differences among groups. However, Sol AspAP and Sol pGluAP progressively decreased with the degree of saturation of the fatty acid used in the diet. In contrast, M-B GluAP progressively increased with the degree of saturation of the fatty acid used in the diet. For M-B AspAP activity, mice fed diets containing FO showed significantly higher levels than those fed diets containing SFO, OO and L but not those containing CO. For M-B pGluAP activity, the highest levels were observed for mice fed diets containing FO and OO. The present data suggest that the type of fat used in the diet may influence the autocrine/paracrine functions of locally synthesized angiotensin peptides and TRH in the testis

  13. Development of pre-implantation porcine embryos cultured within a three-dimensional alginate hydrogel system either conjugated with Arg-Gly-Asp (RGD) peptide or supplemented with secreted phosphoprotein 1 (SPP1)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many uterine specific factors have been shown to be increased within the uterine milieu as the porcine embryo initiates elongation. Secreted phosphoprotein 1 (SPP1) is increased during this time and contains an Arg-Gly-Asp (RGD) peptide sequence that has been shown to bind to cell surface integrins ...

  14. A new hemoglobin variant: Hb Meylan [β73(E17)Asp → Phe; HBB: c.220G>T; c.221A>T] with a double base mutation at the same codon.

    PubMed

    Renoux, Céline; Feray, Cécile; Joly, Philippe; Zanella-Cleon, Isabelle; Garcia, Caroline; Lacan, Philippe; Couprie, Nicole; Francina, Alain

    2015-01-01

    We report a new β-globin chain variant: Hb Meylan [β73(E17)Asp → Phe; HBB: c.220G>T; c.221A>T]. The new variant results from a double nucleotide mutation at the same codon. The possible molecular mechanisms are discussed.

  15. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  16. Mechanism of a GatCAB amidotransferase: aspartyl-tRNA synthetase increases its affinity for Asp-tRNA(Asn) and novel aminoacyl-tRNA analogues are competitive inhibitors.

    PubMed

    Huot, Jonathan L; Balg, Christian; Jahn, Dieter; Moser, Jürgen; Emond, Audrey; Blais, Sébastien P; Chênevert, Robert; Lapointe, Jacques

    2007-11-13

    The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.

  17. Effect of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of A{beta}{sub 20-29} peptides

    SciTech Connect

    Shanmugam, Ganesh; Polavarapu, Prasad L. . E-mail: Prasad.L.Polavarapu@Vanderbilt.edu; Hallgas, Balazs; Majer, Zsuzsa

    2005-09-30

    The effects of D-amino acids at Asp{sup 23} and Ser{sup 26} residues on the conformational preference of {beta}-amyloid (A{beta}) peptide fragment (A{beta}{sub 20-29}) have been studied using different spectroscopic techniques, namely vibrational circular dichroism (VCD), vibrational absorption, and electronic circular dichroism. To study the structure of the A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides under different conditions, the spectra were measured in 10 mM acetate buffer (pH 3) and in 2,2,2-trifluoroethanol (TFE). The spectroscopic results indicated that at pH 3, A{beta}{sub 20-29} peptide takes random coil with {beta}-turn structure, while [D-Ser{sup 26}]A{beta}{sub 20-29} peptide adopts significant amount of polyproline II (PPII) type structure along with {beta}-turn contribution and D-Asp-substituted peptide ([D-Asp{sup 23}]A{beta}{sub 20-29}) adopts predominantly PPII type structure. The increased propensity for PPII conformation upon D-amino acid substitution, in acidic medium, has important biological implications. In TFE, A{beta}{sub 20-29}, [D-Asp{sup 23}]A{beta}{sub 20-29}, and [D-Ser{sup 26}]A{beta}{sub 20-29} peptides adopt 3{sub 10}-helix, {alpha}-helix, and random coil with some {beta}-turn structures, respectively. The VCD data obtained for the A{beta} peptide films suggested that the secondary structures for the peptide films are not the same as those for corresponding solution and are also different among the A{beta} peptides studied here. This observation suggests that dehydration can have a significant influence on the structural preferences of these peptides.

  18. A novel adenosine A(1) and A(2A) receptor antagonist ASP5854 ameliorates motor impairment in MPTP-treated marmosets: comparison with existing anti-Parkinson's disease drugs.

    PubMed

    Mihara, Takuma; Iwashita, Akinori; Matsuoka, Nobuya

    2008-12-12

    Recent evidence indicates that adenosine A(2A) receptor antagonists hold therapeutic potential for the treatment of Parkinson's disease (PD). A study on the novel adenosine A(1) and A(2A) receptor dual antagonist 5-[5-amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854) showed it to be effective in various rodents models of PD and cognition. In the present study, we further investigated the potential of ASP5854 as an anti-PD drug using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets, which is a highly predictive model of clinical efficacy in PD, and compared its effect with those of existing anti-PD drugs. ASP5854 significantly and dose-dependently improved the total motor disability score for 7h at doses higher than 1mg/kg, and significantly increased total locomotor activity at doses higher than 0.1mg/kg without adverse effects. l-3,4-Dihydroxyphenylalanine+benserazide and bromocriptine also significantly improved the motor disability score and the hypolocomotion caused by MPTP treatment in a dose-dependent fashion. This amelioration was significant at 32+8 and 10-32 mg/kg, respectively, although bromocriptine induced severe emesis. Trihexiphenidyl also significantly improved the total motor disability score at doses of 10-32 mg/kg; however, while a significant increase in the total locomotor activity was observed at 10mg/kg, the drug induced ataxia-like behavior at 32 mg/kg. On the other hand, neither selegiline nor amantadine improved the total motor disability and hypolocomotion. These data substantiate the evidence that the novel adenosine antagonist ASP5854 exerts comparable anti-PD activity with existing anti-PD drugs, which indicates that ASP5854 might have potential to ameliorate motor deficits in PD.

  19. Evaluation of recombinant Onchocerca volvulus activation associated protein-1 (ASP-1) as a potent Th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines.

    PubMed

    Xiao, Wenjun; Du, Lanying; Liang, Chao; Guan, Jie; Jiang, Shibo; Lustigman, Sara; He, Yuxian; Zhou, Yusen

    2008-09-15

    Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-gamma-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines. PMID:18675867

  20. Affective Dynamics in Psychopathology

    PubMed Central

    Trull, Timothy J.; Lane, Sean P.; Koval, Peter; Ebner-Priemer, Ulrich W.

    2016-01-01

    We discuss three varieties of affective dynamics (affective instability, emotional inertia, and emotional differentiation). In each case, we suggest how these affective dynamics should be operationalized and measured in daily life using time-intensive methods, like ecological momentary assessment or ambulatory assessment, and recommend time-sensitive analyses that take into account not only the variability but also the temporal dependency of reports. Studies that explore how these affective dynamics are associated with psychological disorders and symptoms are reviewed, and we emphasize that these affective processes are within a nexus of other components of emotion regulation.

  1. Principal transcriptional regulation and genome-wide system interactions of the Asp-family and aromatic amino acid networks of amino acid metabolism in plants.

    PubMed

    Less, Hadar; Angelovici, Ruthie; Tzin, Vered; Galili, Gad

    2010-10-01

    Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energy-associated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058-2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156-167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain

  2. The Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin†,‡

    PubMed Central

    D’Antonio, Edward L.; D’Antonio, Jennifer; de Serrano, Vesna; Gracz, Hanna; Thompson, Matthew K.; Ghiladi, Reza A.; Bowden, Edmond F.; Franzen, Stefan

    2011-01-01

    The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine-86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D (PDB 3MYN) and M86E (PDB 3MYM) mutants reveals the structural origins of a stable catalytic triad in DHP A. A decrease in rate of H2O2 activation, as well as a lowered reduction potential than the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine to bind to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is 5-coordinate and this resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a 6-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a 6-coordinate low-spin adduct, which inhibits the ligation and activation of H2O2 as required for peroxidase activity. The results suggest that the role of the proximal charge relay in peroxidases regulates the redox potential of the heme Fe, but that the trans effect is a carefully balanced property that can both activate H2O2 and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, three mutants in the M86 position, M86A, M86D, and M86E were studied by spectroelectrochemistry and NMR spectroscopy of 13C15N -labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the Nδ hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge

  3. QM/MM free-energy simulations of reaction in Serratia marcescens Chitinase B reveal the protonation state of Asp142 and the critical role of Tyr214.

    PubMed

    Jitonnom, Jitrayut; Limb, Michael A L; Mulholland, Adrian J

    2014-05-01

    Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of β-1,4-glycosidic bond, with retention of configuration, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. Here, both elementary steps (glycosylation and deglycosylation) of the ChiB-catalyzed reaction are investigated by means of combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations at the SCC-DFTB/CHARMM22 level of theory. We examine the influence of the Asp142 protonation state on the reaction and the role that this residue performs in the reaction. Our simulations show that reaction with a neutral Asp142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary ((1,4)B↔(4)H5↔(4)C1) adopted by the substrate (bound in subsite -1) along the preferred reaction pathway is also provided by the simulations. The relative energies of the stationary points found along the reaction pathway calculated with SCC-DFTB and B3LYP were compared. The results suggest that SCC-DFTB is an accurate method for estimating the relative barriers for both steps of the reaction; however, it was found to overestimate the relative energy of an intermediate formed in the reaction when compared with the higher level of theory. Glycosylation is suggested to be a rate-determining step in the reaction with calculated overall reaction free-energy barrier of 20.5 kcal/mol, in a reasonable agreement with the 16.1 kcal/mol barrier derived from the experiment. The role of Tyr214 in catalysis was also investigated with the results, indicating that the residue plays a critical role in the deglycosylation step of the reaction. Simulations of the enzyme-product complex were also performed with an unbinding event suggested to have been observed

  4. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    PubMed

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID

  5. Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations.

    PubMed

    Liu, Hui; Wang, Hua-Fang; Tang, Liang; Yang, Yan; Wang, Qing-Yun; Zeng, Wei; Wu, Ying-Ying; Cheng, Zhi-Peng; Hu, Bei; Guo, Tao; Hu, Yu

    2015-05-25

    Hereditary protein C deficiency (PCD) is an autosomal inherited disorder associated with high risk for venous thromboembolism (VTE). This study aimed to explore the functional consequences of two missense mutations, p.Asp297His and p.Val420Ile, responsible for type I/II PCD and recurrent deep vein thrombosis (DVT) in a Chinese family. The plasma protein C activities (PC:A) of the proband and his sister were reduced to 4% and 5% of normal activity. However, protein C antigen (PC:Ag) concentrations were not equally decreased, with levels of 90.5% and 88.7%, respectively. Two missense mutations p.Asp297His and p.Val420Leu were identified in the protein C gene (PROC). The PC:A and PC:Ag levels in heterozygous state for p.Asp297His were 66% and 64.8%, whereas in heterozygous state for p.Val420Leu, these levels were 67% and 145%, respectively. Wild type (WT) and two mutant PROC cDNA expression plasmids were constructed and transfected into HEK 293T cells. Western blot analysis revealed that both p.Asp297His and p.Val420Leu showed a normal intracellular protein level. The extracellular protein level and specific activity of p.Asp297His were equally reduced to 37.7 ± 4.3% and 22.1 ± 2.5%, respectively. Mutant p.Val420Leu showed a relatively higher PC:Ag level and undetectable PC:A. Immunofluorescence staining revealed that WT and p.Val420Leu proteins were largely co-localized with both the protein disulfide isomerase (PDI) and cis-Golgi Marker (GM130), while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130. The thrombosis symptom in this family was associated with the two missense mutations in the PROC gene.

  6. Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297.

    PubMed

    Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

    2014-06-01

    The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies. PMID:24740840

  7. Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico

    PubMed Central

    Cruz-Hipolito, Hugo; Fernandez, Pablo; Alcantara, Ricardo; Gherekhloo, Javid; Osuna, Maria Dolores; De Prado, Rafael

    2015-01-01

    Herbicides that inhibit acetyl coenzyme A carboxylase (ACCase) are commonly used in Mexico to control weedy grasses such as little seed canarygrass (Phalaris minor). These herbicides are classified into three major families (ariloxyphenoxypropionates (APP), cyclohexanodiones (CHD), and, recently, phenylpyrazolines (PPZ)). In this work, the resistance to ACCase (APP, CHD, and PPZ) inhibiting herbicides was studied in a biotype of Phalaris minor (P. minor) from Mexico, by carrying out bioassays at the whole-plant level and investigating the mechanism behind this resistance. Dose-response and ACCase in vitro activity assays showed cross-resistance to all ACCase herbicides used. There was no difference in the absorption, translocation, and metabolism of the 14C-diclofop-methyl between the R and S biotypes. The PCR generated CT domain fragments of ACCase from the R biotype and an S reference were sequenced and compared. The Ile-1781-Leu and Asp-2078-Gly point mutations were identified. These mutations could explain the loss of affinity for ACCase by the ACCase-inhibing herbicides. This is the first report showing that this substitution confers resistance to APP, CHD, and PPZ herbicides in P. minor from Mexico. The mutations have been described previously only in a few cases; however, this is the first study reporting on a pattern of cross-resistance with these mutations in P. minor. The findings could be useful for better management of resistant biotypes carrying similar mutations. PMID:26370967

  8. Repetitive Gly-Leu-Lys-Gly-Glu-Asn-Arg-Gly-Asp peptide derived from collagen and fibronectin for improving cell-scaffold interaction.

    PubMed

    Chaisri, Patcharaporn; Chingsungnoen, Artit; Siri, Sineenat

    2015-03-01

    Suitable scaffolds for tissue engineering should provide a microenvironment for cell dwelling and directing cell behavior that resemble the native environment. Three-dimensional geometry of electrospun scaffolds well supports cell deposition, but they often lack biomacromolecules to induce cell responses. In this work, the repetitive collagen and fibronectin motif (rCF) peptide containing multiple repeats of Gly-Leu-Lys-Gly-Glu-Asn-Arg-Gly-Asp sequence derived from the cell adhesion motifs of collagen and fibronectin was produced as the alternative agent to induce cell-scaffold interaction. The DNA fragment encoding rCF peptide was amplified by a polymerase chain reaction using overlap primers without a DNA template, cloned into a protein expression vector, and expressed as a His-tag fusion peptide in Escherichia coli. The purified rCF peptide possessed cell adhesion activity about 1.5-fold of the commercial RGD peptide. The rCF peptide was grafted onto the electrospun PCL scaffold via RF plasma of Ar/O2 discharge and acrylic acid treatment. The immobilized rCF peptide significantly increased surface hydrophilicity and enhanced cell proliferation of the electrospun PCL scaffold. These findings suggest the potential application of rCF peptide for improving the biomimetic functions of polymeric scaffolds for tissue engineering.

  9. The use of synthetic analogues of Arg-Gly-Asp (RGD) and soluble receptor of tumor necrosis factor to prevent acute and chronic experimental liver injury.

    PubMed

    Bruck, R; Hershkoviz, R; Lider, O; Shirin, H; Aeed, H; Halpern, Z

    1997-01-01

    In chronic viral hepatitis, autoimmune hepatitis, and some chronic cholestatic liver diseases, T-lymphocytes serve as effector cells of the immunostimulatory processes. Cellular interactions of immune cells with extracellular matrix (ECM) components are regulated primarily via the beta 1 subfamily of integrin receptors. The target epitope of several such integrin receptors is the Arg-Gly-Asp (RGD) sequence, a cell adhesion motif shared by several matrix-associated adhesive glycoproteins. We review the use of synthetic nonpeptidic analogues of RGD and of soluble receptor of tumor necrosis factor (TNF)-alpha in the prevention of immune-mediated, concanavalin A-induced liver damage in mice and of RGD analogues in inhibiting the development of liver cirrhosis in rats. The concanavalin A-induced elevation of serum transaminases and TNF-alpha, and the infiltration of liver tissue by inflammatory cells, were inhibited by pretreatment of the mice with the synthetic RGD mimetics and soluble TNF receptor. In rats, the progression of thioacetamide-induced liver cirrhosis was markedly inhibited by the coadministration of the RGD mimetic SF-6,5. The compounds described here may be examined therapeutically for pathological conditions in the liver, manifested as necroinflammation, cholestasis and fibrosis. PMID:9626759

  10. Rational improvement of gp41-targeting HIV-1 fusion inhibitors: an innovatively designed Ile-Asp-Leu tail with alternative conformations

    PubMed Central

    Zhu, Yun; Su, Shan; Qin, Lili; Wang, Qian; Shi, Lei; Ma, Zhenxuan; Tang, Jianchao; Jiang, Shibo; Lu, Lu; Ye, Sheng; Zhang, Rongguang

    2016-01-01

    Peptides derived from the C-terminal heptad repeat (CHR) of HIV gp41 have been developed as effective fusion inhibitors against HIV-1, but facing the challenges of enhancing potency and stability. Here, we report a rationally designed novel HIV-1 fusion inhibitor derived from CHR-derived peptide (Trp628~Gln653, named CP), but with an innovative Ile-Asp-Leu tail (IDL) that dramatically increased the inhibitory activity by up to 100 folds. We also determined the crystal structures of artificial fusion peptides N36- and N43-L6-CP-IDL. Although the overall structures of both fusion peptides share the canonical six-helix bundle (6-HB) configuration, their IDL tails adopt two different conformations: a one-turn helix with the N36, and a hook-like structure with the longer N43. Structural comparison showed that the hook-like IDL tail possesses a larger interaction interface with NHR than the helical one. Further molecular dynamics simulations of the two 6-HBs and isolated CP-IDL peptides suggested that hook-like form of IDL tail can be stabilized by its binding to NHR trimer. Therefore, CP-IDL has potential for further development as a new HIV fusion inhibitor, and this strategy could be widely used in developing artificial fusion inhibitors against HIV and other enveloped viruses. PMID:27666394

  11. Radiolabeling of a cyclic RGD (cyclo Arg-Gly-Asp-d-Tyr-Lys) peptide using sodium hypochlorite as an oxidizing agent.

    PubMed

    Doll, Stephanie; Woolum, Karen; Kumar, Krishan

    2016-09-01

    A simple and rapid nonradioactive iodide labeling/radiolabeling method for peptides, using an inexpensive oxidizing agent such as sodium hypochlorite and a cyclic peptide, cRGDyK (cyclo Arg-Gly-Asp-d-Tyr-Lys), was developed in this work. Labeling reaction was optimized by conducting experiments under variable ratios of the reagents, the reaction times, and the pH. The study demonstrated that radiolabeling of the cyclic peptide was fast and pH independent. Monoiodinated and di-iodinated cRGDyK were formed under all conditions and varied with the ratio of the reagents and the reaction time. Total percent of the iodinated cRGDyK (monoiodinated and di-iodinated cRGDyK) varied between 44 and 100 depending on the reaction conditions. Excess cyclic peptide over equal molar ratio of sodium iodide and sodium hypochlorite yielded in predominant amounts of monoiodinated cRGDyK, ie, >60% under 2:1:1 ratio and ~88% under 5:1:1 ratio of cRGDyK:sodium iodide:sodium hypochlorite. PMID:27577980

  12. A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma.

    PubMed Central

    Kennan, A M; Mansergh, F C; Fingert, J H; Clark, T; Ayuso, C; Kenna, P F; Humphries, P; Farrar, G J

    1998-01-01

    Glaucoma describes a clinically and genetically heterogeneous group of diseases that result in optic neuropathy and progressive loss of visual fields. A gene for juvenile onset primary open angle glaucoma JOAG) has recently been mapped to 1q21-31. Mutations in the trabecular meshwork induced glucocorticoid response gene (TIGR, also known as myocilin or the GLC1A locus) have been found to cause both juvenile and later onset primary open angle glaucoma. Family TCD-POAG1 is a Spanish kindred, which segregates JOAG in an autosomal dominant fashion. This family was found to be linked to the previously identified GLC1A locus on chromosome 1q. Direct sequencing of the TIGR/myocilin gene showed a heterozygous A to C transition in codon 380, resulting in the substitution of alanine for aspartic acid (Asp380Ala). This substitution created a StyI restriction site, which segregated with the JOAG phenotype and permitted rapid screening of all members of the family. This restriction site was not present in 60 controls. Images PMID:9832047

  13. Covalent attachment of cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) to poly(etheretherketone) surface by tailored silanization layers technique

    NASA Astrophysics Data System (ADS)

    Zheng, Yanyan; Xiong, Chengdong; Li, Xiaoyu; Zhang, Lifang

    2014-11-01

    Poly(etheretherketone) (PEEK) is a rigid semicrystalline polymer that combines excellent mechanical properties, broad chemical resistance and bone-like stiffness and is widely used in biomedical fields. However, PEEK is naturally bioinert, leading to limited biomedical applications, especially when a direct bone-implant osteointegration is desired. In this study, a three-step reaction procedure was employed to immobilize the cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) on the surface of PEEK sheet by covalent chemical attachment to favor cell adhesion and proliferation. First, hydroxylation-pretreated PEEK surfaces were silanized with 7-Oct-1-enyltrichlorosilane (OETS) in dry cyclohexane, resulting in a silanization layer with terminal ethenyl. Second, the terminal ethylenic double bonds of the silanization layer on PEEK surface were converted to carboxyl groups through acidic potassium manganate oxidation. Finally, GRGD was covalently attached by carbodiimide mediated condensation between the carboxyl on PEEK surface and amine presents in GRGD. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, surface profiler and water contact angle measurements were applied to characterize the modified surfaces. The effect of cells attachment and proliferation on each specimen was investigated. Pre-osteoblast cells (MC3T3-E1) attachment, spreading and proliferation were improved effectively on GRGD-modified PEEK surface. PEEK modified with GRGD on its surface has potential use in orthopedic or dental implants.

  14. Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297.

    PubMed

    Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

    2014-06-01

    The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies.

  15. Affectional Patterns of Adolescents.

    ERIC Educational Resources Information Center

    O'Donnell, William J.

    1979-01-01

    This study sought to determine if there is a shift with age in affection (1) from parents to friends, (2) from one parent to the other, and (3) from same-sex to opposite-sex friends. Subjects, eighth graders and eleventh graders, completed the Measurement of Family Affective Structure. (Author)

  16. Affective Involvement Instrument.

    ERIC Educational Resources Information Center

    Lemlech, Johanna K.

    1970-01-01

    The Affective Involvement Instrument (AII) describes and classifies affective involvement in the process of decision-making as it occurs during classroom activities such as role-playing or group discussions. The thirty-celled instrument behaviorizes the six processes involved in decision-making and combines them with the taxonomic levels of the…

  17. [Affect and mimetic behavior].

    PubMed

    Zepf, S; Ullrich, B; Hartmann, S

    1998-05-01

    The relationship between facial expression and experienced affect presents many problems. The two diametrically opposed positions proposing solutions to this problem are exemplified using the conceptions of Mandler u. Izard. The underlying premises of both conceptions still prevail in various forms. The authors reject the concepts according to which facial expression is merely correlated to the affects (see Mandler 1975) as well as the view that facial expression controls the affects (see Izard 1977). The relationship between affect and facial expression is reexamined, subjecting it to a semiotic, essentially semantic analysis similar to the Ogden and Richards' language and meaning approach. This analysis involves a critical discussion of Scherer's attempt of a purely communicational interpretation using Bühler's organon model. In the author's approach, facial expression is seen not simply as a system of signals, but as a system of representative signs which signify the affects and refer to the emotive meaning of things for the subject. The authors develop the thesis that human beings are not born simply with the ability to speak, but also with the abstract possibility of performing facial expressions. This ability develops by way of coordinating patterns of expressions, which are presumably phylogenetically determined, with affects that take on a socially determined individual form, similar to language acquisition during socialisation. The authors discuss the methodological implications arising for studies investigating the affective meaning of facial expressions. PMID:9632951

  18. Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure

    NASA Technical Reports Server (NTRS)

    Sasaki, J.; Shichida, Y.; Lanyi, J. K.; Maeda, A.

    1992-01-01

    The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferle, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.

  19. Compounds affecting cholesterol absorption

    NASA Technical Reports Server (NTRS)

    Hua, Duy H. (Inventor); Koo, Sung I. (Inventor); Noh, Sang K. (Inventor)

    2004-01-01

    A class of novel compounds is described for use in affecting lymphatic absorption of cholesterol. Compounds of particular interest are defined by Formula I: ##STR1## or a pharmaceutically acceptable salt thereof.

  20. In situ tracer tests to determine retention properties of a block scale fracture network in granitic rock at the Aspö Hard Rock Laboratory, Sweden.

    PubMed

    Andersson, Peter; Byegård, Johan; Tullborg, Eva-Lena; Doe, Thomas; Hermanson, Jan; Winberg, Anders

    2004-06-01

    Experiments were conducted at the Aspö Hard Rock Laboratory in order to improve the understanding of radionuclide retention properties of fractured crystalline bedrock in the 10-100 m scale (TRUE Block Scale Project, jointly funded by ANDRA, ENRESA, Nirex, JNC, Posiva and SKB). A series of tracer experiments were performed using sorbing tracers in three different flow paths. The different flow paths had Euclidian lengths of 14, 17 and 33 m, respectively, and one to three water conducting structures. Four tests were performed using different cocktails made up of radioactive sorbing tracers (22,24Na+, 42K+, 47Ca2+, 85Sr2+, 83,86Rb+, 131,133Ba2+ and 134,137Cs+). For each tracer injection, the breakthrough of sorbing tracers was compared to the breakthrough of a conservative tracer, 82Br-, 131I-, HTO and 186ReO4-, respectively. In the two longer flow paths, no breakthrough of 83Rb+ and 137Cs+ was observed after 8 months of pumping. Selected tracer tests were subject to basic modelling in which a one-dimensional (1D) advection-dispersion model, including surface sorption, and an unlimited matrix diffusion were used for the interpretation of the results. The results of the modelling indicated that there is a slightly higher mass transfer into a highly porous material in the block-scale experiment compared with in situ experiments performed over shorter distances and significantly higher than what would have been expected from laboratory data obtained from studies of the interactions in nonaltered intact rock.

  1. Evaluation of Osteoblast-Like Cell Viability and Differentiation on the Gly-Arg-Gly-Asp-Ser Peptide Immobilized Titanium Dioxide Nanotube via Chemical Grafting.

    PubMed

    Kim, Ga-Hyun; Kim, Il-Shin; Park, Sang-Won; Lee, Kwangmin; Yun, Kwi-Dug; Kim, Hyun-Seung; Oh, Gye-Jeong; Ji, Min-Kyung; Lim, Hyun-Pil

    2016-02-01

    This study examined the effect of the immobilization of the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide on titanium dioxide (TiO2) nanotube via chemical grafting on osteoblast-like cell (MG-63) viability and differentiation. The specimens were divided into two groups; TiO2 nanotubes and GRGDS-immobilized TiO2 nanotubes. The surface characteristics of GRGDS-immobilized TiO2 nanotubes were observed by using X-ray photoelectron spectroscopy (XPS) and a field emission scanning electron microscope (FE-SEM). The morphology of cells on specimens was observed by FE-SEM after 2 hr and 24 hr. The level of cell viability was investigated via a tetrazolium (XTT) assay after 2 and 4 days. Alkaline phosphatase (ALP) activity was evaluated to measure the cell differentiation after 4 and 7 days. The presence of nitrogen up-regulation or C==O carbons con- firmed that TiO2 nanotubes were immobilized with GRGDS peptides. Cell adhesion was enhanced on the GRGDS-immobilized TiO2 nanotubes compared to TiO2 nanotubes. Furthermore, significantly increased cell spreading and proliferation were observed with the cells grown on GRGDS-immobilized TiO2 nanotubes (P < .05). However, there was no significant difference in ALP activity between GRGDS-immobilized TiO2 nanotubes and TiO2 nanotubes. These results suggest that the GRGDS-immobilized TiO2 nanotubes might be effective in improving the osseointegration of dental implants.

  2. Specific and global coagulation tests in patients with mild haemophilia A with a double mutation (Glu113Asp, Arg593Cys)

    PubMed Central

    Bakija, Alenka Trampuš; Debeljak, Maruša; Zupan, Irena Preložnik; Dolničar, Majda Benedik; Kovač, Jernej; Jazbec, Janez

    2015-01-01

    Background Heterogeneous bleeding phenotypes are observed in haemophilia A patients with the same mutation in the F8 gene. Specific mutations in the A2 domain of factor VIII are associated with mild haemophilia and a higher risk of inhibitor development. Double mutations in mild haemophilia A are rarely reported. In this study, we investigated the in vitro function of factor VIII, performing different specific and global coagulation assays, observed clinical characteristics and assessed the possible predictive diagnostic value of the differences. Materials and methods The clinical features of haemophiliacs with a mild phenotype were reviewed. Blood samples were obtained and analysed for mutations and coagulation assays: activated partial thromboplastin time, one-stage and chromogenic factor VIII activity, factor VIII antigen and rotational thromboelastometry. Results We report on a cohort of 22 patients with double Glu113Asp, Arg593Cys mutations. All our patients have a quantitative defect of factor VIII and preserved similar functional activity. Factor VIII activities measured by the one-stage or chromogenic method were not discrepant, although the chromogenic assay resulted in 20% lower factor VIII activities. Waveform analysis showed a lower maximum value of the second derivative curve (Max2) of APTT with curve shape alternation, while thromboelastometry (INTEM) showed low sensitivity in comparison to results in a normal population. Discussion In genotyping, the coexistence of a second mutation should never be excluded, especially in cases of discordant clinical presentation. Waveform analysis correlates better with factor VIII activity than thromboelastometry and the Max2 parameter could provide additional information in managing haemophilia patients. The utility of specific factor activity and global haemostatic assays in general practice still needs to be investigated. PMID:26057490

  3. Evaluation of Osteoblast-Like Cell Viability and Differentiation on the Gly-Arg-Gly-Asp-Ser Peptide Immobilized Titanium Dioxide Nanotube via Chemical Grafting.

    PubMed

    Kim, Ga-Hyun; Kim, Il-Shin; Park, Sang-Won; Lee, Kwangmin; Yun, Kwi-Dug; Kim, Hyun-Seung; Oh, Gye-Jeong; Ji, Min-Kyung; Lim, Hyun-Pil

    2016-02-01

    This study examined the effect of the immobilization of the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide on titanium dioxide (TiO2) nanotube via chemical grafting on osteoblast-like cell (MG-63) viability and differentiation. The specimens were divided into two groups; TiO2 nanotubes and GRGDS-immobilized TiO2 nanotubes. The surface characteristics of GRGDS-immobilized TiO2 nanotubes were observed by using X-ray photoelectron spectroscopy (XPS) and a field emission scanning electron microscope (FE-SEM). The morphology of cells on specimens was observed by FE-SEM after 2 hr and 24 hr. The level of cell viability was investigated via a tetrazolium (XTT) assay after 2 and 4 days. Alkaline phosphatase (ALP) activity was evaluated to measure the cell differentiation after 4 and 7 days. The presence of nitrogen up-regulation or C==O carbons con- firmed that TiO2 nanotubes were immobilized with GRGDS peptides. Cell adhesion was enhanced on the GRGDS-immobilized TiO2 nanotubes compared to TiO2 nanotubes. Furthermore, significantly increased cell spreading and proliferation were observed with the cells grown on GRGDS-immobilized TiO2 nanotubes (P < .05). However, there was no significant difference in ALP activity between GRGDS-immobilized TiO2 nanotubes and TiO2 nanotubes. These results suggest that the GRGDS-immobilized TiO2 nanotubes might be effective in improving the osseointegration of dental implants. PMID:27433593

  4. Studies on the influence of combustion exhaust gases and the products of their reaction with ammonia on the living organism. II. The influence on aspartate aminotransferase (AspAT) and alanine aminotransferase (AiAt) activities in the liver of guinea pig.

    PubMed

    Lewandowska-Tokarz, A; Stanosek, J; Ludyga, K; Kochanski, L

    1981-01-01

    The behaviour of aspartate aminotransferase (AspAT) an alanine aminotransferase (AIAT) in the whole homogenate and subcellular liver fractions of guinea pigs exposed to combustion exhaust gases and the neutralization products of these gases is presented in this paper. In the liver of animals exposed to the chronic action of combustion exhaust gases a decrease of both enzyme activities in the whole homogenate as well as in the subcellular fractions could be noted. Statistically significant changes are shown by AspAT. In the group of animals subjected to the action of neutralization products an increase of AIAT activity was observed. The activity of AspAT still shows a decrease, but less distinct in comparison with group I. An exception here is the mitochondrial fraction in which the AspAT activity is distinctly increased.

  5. Affective responses to dance.

    PubMed

    Christensen, Julia F; Pollick, Frank E; Lambrechts, Anna; Gomila, Antoni

    2016-07-01

    The objective of the present work was the characterization of mechanisms by which affective experiences are elicited in observers when watching dance movements. A total of 203 dance stimuli from a normed stimuli library were used in a series of independent experiments. The following measures were obtained: (i) subjective measures of 97 dance-naïve participants' affective responses (Likert scale ratings, interviews); and (ii) objective measures of the physical parameters of the stimuli (motion energy, luminance), and of the movements represented in the stimuli (roundedness, impressiveness). Results showed that (i) participants' ratings of felt and perceived affect differed, (ii) felt and perceived valence but not arousal ratings correlated with physical parameters of the stimuli (motion energy and luminance), (iii) roundedness in posture shape was related to the experience of more positive emotion than edgy shapes (1 of 3 assessed rounded shapes showed a clear effect on positiveness ratings while a second reached trend level significance), (iv) more impressive movements resulted in more positive affective responses, (v) dance triggered affective experiences through the imagery and autobiographical memories it elicited in some people, and (vi) the physical parameters of the video stimuli correlated only weakly and negatively with the aesthetics ratings of beauty, liking and interest. The novelty of the present approach was twofold; (i) the assessment of multiple affect-inducing mechanisms, and (ii) the use of one single normed stimulus set. The results from this approach lend support to both previous and present findings. Results are discussed with regards to current literature in the field of empirical aesthetics and affective neuroscience.

  6. Affective responses to dance.

    PubMed

    Christensen, Julia F; Pollick, Frank E; Lambrechts, Anna; Gomila, Antoni

    2016-07-01

    The objective of the present work was the characterization of mechanisms by which affective experiences are elicited in observers when watching dance movements. A total of 203 dance stimuli from a normed stimuli library were used in a series of independent experiments. The following measures were obtained: (i) subjective measures of 97 dance-naïve participants' affective responses (Likert scale ratings, interviews); and (ii) objective measures of the physical parameters of the stimuli (motion energy, luminance), and of the movements represented in the stimuli (roundedness, impressiveness). Results showed that (i) participants' ratings of felt and perceived affect differed, (ii) felt and perceived valence but not arousal ratings correlated with physical parameters of the stimuli (motion energy and luminance), (iii) roundedness in posture shape was related to the experience of more positive emotion than edgy shapes (1 of 3 assessed rounded shapes showed a clear effect on positiveness ratings while a second reached trend level significance), (iv) more impressive movements resulted in more positive affective responses, (v) dance triggered affective experiences through the imagery and autobiographical memories it elicited in some people, and (vi) the physical parameters of the video stimuli correlated only weakly and negatively with the aesthetics ratings of beauty, liking and interest. The novelty of the present approach was twofold; (i) the assessment of multiple affect-inducing mechanisms, and (ii) the use of one single normed stimulus set. The results from this approach lend support to both previous and present findings. Results are discussed with regards to current literature in the field of empirical aesthetics and affective neuroscience. PMID:27235953

  7. Lack of linkage between the corticotropin-releasing hormone (CRH) gene and bipolar affective disorder.

    PubMed

    Stratakis, C A; Sarlis, N J; Berrettini, W H; Badner, J A; Chrousos, G P; Gershon, E S; Detera-Wadleigh, S D

    1997-01-01

    Corticotropin-releasing hormone (CRH) plays a key role in the regulation of the stress response. Abnormalities in CRH secretion have been documented in both the depression and manic phases of bipolar disorder (BPD). In the present study, we investigated genetic linkage between the CRH gene and BPD in 22 pedigrees. A highly informative, short tandem repeat (STR) polymorphism adjacent to the CRH gene on human chromosomal region 8q13 was used to examine linkage. Affected sibling pair (ASP) and the likelihood-based disequilibrium tests revealed nonsignificant values. We conclude that the CRH gene is not linked to BPD; if genes involved in the regulation of stress response are indeed linked to BPD, the search should be directed towards those that regulate CRH secretion or its effects on target tissues.

  8. Elements affecting runway traction

    NASA Technical Reports Server (NTRS)

    Horne, W. B.

    1974-01-01

    The five basic elements affecting runway traction for jet transport aircraft operation are identified and described in terms of pilot, aircraft system, atmospheric, tire, and pavement performance factors or parameters. Where possible, research results are summarized, and means for restoring or improving runway traction for these different conditions are discussed.

  9. What Variables Affect Solubility?

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn

    2003-01-01

    Helps middle school students understand the concept of solubility through hands-on experience with a variety of liquids and solids. As they explore factors that affect solubility and saturation, students gain content mastery and an understanding of the inquiry process. Also enables teachers to authentically assess student performance on several…

  10. How Body Affects Brain.

    PubMed

    Suzuki, Wendy A

    2016-08-01

    Studies show that physical exercise can affect a range of brain and cognitive functions. However, little is known about the peripheral signals that initiate these central changes. Moon et al. (2016) provide exciting new evidence that a novel myokine, cathepsin B (CTSB), released with exercise is associated with improved memory. PMID:27508865

  11. Food Affects Human Behavior.

    ERIC Educational Resources Information Center

    Kolata, Gina

    1982-01-01

    A conference on whether food and nutrients affect human behavior was held on November 9, 1982 at the Massachusetts Institute of Technology. Various research studies on this topic are reviewed, including the effects of food on brain biochemistry (particularly sleep) and effects of tryptophane as a pain reducer. (JN)

  12. Factors affecting soil cohesion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil erodibility is a measure of a soil’s resistance against erosive forces and is affected by both intrinsic (or inherent) soil property and the extrinsic condition at the time erodibility measurement is made. Since soil erodibility is usually calculated from results obtained from erosion experimen...

  13. Affective antecedents of revenge.

    PubMed

    O'Connor, Kieran; Adams, Gabrielle S

    2013-02-01

    We propose that revenge responses are often influenced more by affective reactions than by deliberate decision making as McCullough et al. suggest. We review social psychological evidence suggesting that justice judgments and reactions may be determined more by emotions than by cognitions.

  14. Solute transport in crystalline rocks at Aspö--II: blind predictions, inverse modelling and lessons learnt from test STT1.

    PubMed

    Jakob, Andreas; Mazurek, Martin; Heer, Walter

    2003-03-01

    Based on the results from detailed structural and petrological characterisation and on up-scaled laboratory values for sorption and diffusion, blind predictions were made for the STT1 dipole tracer test performed in the Swedish Aspö Hard Rock Laboratory. The tracers used were nonsorbing, such as uranine and tritiated water, weakly sorbing 22Na(+), 85Sr(2+), 47Ca(2+)and more strongly sorbing 86Rb(+), 133Ba(2+), 137Cs(+). Our model consists of two parts: (1) a flow part based on a 2D-streamtube formalism accounting for the natural background flow field and with an underlying homogeneous and isotropic transmissivity field and (2) a transport part in terms of the dual porosity medium approach which is linked to the flow part by the flow porosity. The calibration of the model was done using the data from one single uranine breakthrough (PDT3). The study clearly showed that matrix diffusion into a highly porous material, fault gouge, had to be included in our model evidenced by the characteristic shape of the breakthrough curve and in line with geological observations. After the disclosure of the measurements, it turned out that, in spite of the simplicity of our model, the prediction for the nonsorbing and weakly sorbing tracers was fairly good. The blind prediction for the more strongly sorbing tracers was in general less accurate. The reason for the good predictions is deemed to be the result of the choice of a model structure strongly based on geological observation. The breakthrough curves were inversely modelled to determine in situ values for the transport parameters and to draw consequences on the model structure applied. For good fits, only one additional fracture family in contact with cataclasite had to be taken into account, but no new transport mechanisms had to be invoked. The in situ values for the effective diffusion coefficient for fault gouge are a factor of 2-15 larger than the laboratory data. For cataclasite, both data sets have values comparable to

  15. Solute transport in crystalline rocks at Aspö--II: blind predictions, inverse modelling and lessons learnt from test STT1.

    PubMed

    Jakob, Andreas; Mazurek, Martin; Heer, Walter

    2003-03-01

    Based on the results from detailed structural and petrological characterisation and on up-scaled laboratory values for sorption and diffusion, blind predictions were made for the STT1 dipole tracer test performed in the Swedish Aspö Hard Rock Laboratory. The tracers used were nonsorbing, such as uranine and tritiated water, weakly sorbing 22Na(+), 85Sr(2+), 47Ca(2+)and more strongly sorbing 86Rb(+), 133Ba(2+), 137Cs(+). Our model consists of two parts: (1) a flow part based on a 2D-streamtube formalism accounting for the natural background flow field and with an underlying homogeneous and isotropic transmissivity field and (2) a transport part in terms of the dual porosity medium approach which is linked to the flow part by the flow porosity. The calibration of the model was done using the data from one single uranine breakthrough (PDT3). The study clearly showed that matrix diffusion into a highly porous material, fault gouge, had to be included in our model evidenced by the characteristic shape of the breakthrough curve and in line with geological observations. After the disclosure of the measurements, it turned out that, in spite of the simplicity of our model, the prediction for the nonsorbing and weakly sorbing tracers was fairly good. The blind prediction for the more strongly sorbing tracers was in general less accurate. The reason for the good predictions is deemed to be the result of the choice of a model structure strongly based on geological observation. The breakthrough curves were inversely modelled to determine in situ values for the transport parameters and to draw consequences on the model structure applied. For good fits, only one additional fracture family in contact with cataclasite had to be taken into account, but no new transport mechanisms had to be invoked. The in situ values for the effective diffusion coefficient for fault gouge are a factor of 2-15 larger than the laboratory data. For cataclasite, both data sets have values comparable to

  16. Light-induced movement of the LOV2 domain in an Asp720Asn mutant LOV2-kinase fragment of Arabidopsis phototropin 2.

    PubMed

    Takayama, Yuki; Nakasako, Masayoshi; Okajima, Koji; Iwata, Aya; Kashojiya, Sachiko; Matsui, Yuka; Tokutomi, Satoru

    2011-02-22

    Phototropin, a blue-light receptor protein of plants, triggers phototropic responses, chloroplast relocation, and opening of stomata to maximize the efficiency of photosynthesis. Phototropin is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) that absorb blue light and a serine/theroine kinase domain responsible for light-dependent autophosphorylation leading to cellular signaling cascades. Although the light-activated LOV2 domain is primarily responsible for subsequent activation of the kinase domain, it is unclear how conformational changes in the former transmit to the latter. To understand this molecular mechanism in Arabidopsis phototropin 2, we performed small-angle X-ray scattering analysis on a fragment composed of the LOV2 and kinase domains, which contained an Asp720Asn mutation that led to an absence of ATP binding activity. The scattering data were collected up to a resolution of 25 Å. The apparent molecular weight of the fragment estimated from scattering intensities demonstrated that the fragment existed in a monomeric form in solution. The fragment exhibited photoreversible changes in the scattering profiles, and the radii of gyration under dark and blue-light irradiation conditions were 32.4 and 34.8 Å, respectively. In the dark, the molecular shape restored from the scattering profile appeared as an elongated shape of 110 Å in length and 45 Å in width. The homology modeled LOV2 and kinase domains could be fitted to the molecular shape and appeared to make slight contact. However, under blue-light irradiation, a more extended molecular shape was observed. The changes in the molecular shape and radius of gyration were interpreted as a light-dependent positional shift of the LOV2 domain of approximately 13 Å from the kinase domain. Because the region connecting the LOV2 and kinase domains was categorized as a naturally unfolded polypeptide, we propose that the light-activated LOV2 domain triggers conformational changes in the

  17. Dynamic Synchronization of Teacher-Students Affection in Affective Instruction

    ERIC Educational Resources Information Center

    Zhang, Wenhai; Lu, Jiamei

    2011-01-01

    Based on Bower's affective network theory, the article links the dynamic analysis of affective factors in affective instruction, and presents affective instruction strategic of dynamic synchronization between teacher and students to implement the best ideal mood that promotes students' cognition and affection together. In the process of teaching,…

  18. Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile(33), Ser(34), Asp(37), His(137) and Lys(291).

    PubMed

    Sabui, Subrata; Subramanian, Veedamali S; Kapadia, Rubina; Said, Hamid M

    2016-08-01

    Thiamin plays a critical role in cellular energy metabolism. Mammalian cells obtain the vitamin from their surroundings, converted it to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria via a carrier-mediated process that involves the MTPPT (product of the SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the human MTPPT (hMTPPT), but less is known about structural features that are important for its function. Here, we used a protein-docking model ("Phyre2" and "DockingServer") to predict residues that may be important for function (substrate recognition) of the hMTPPT; we also examined the role of conserved positively-charged residues predicted ("PRALINE") to be in the trans-membrane domains (TMDs) in uptake of the negatively-charged TPP. Among the six residues predicted by the docking model (i.e., Thr(29), Arg(30), Ile(33), Ser(34), Asp(37) and Phe(298)), only Ile(33), Ser(34) and Asp(37) were found to be critical for function. While no change in translational efficiency/protein stability of the Ser(34) mutant was observed, both the Ile(33) and Asp(37) mutants showed a decrease in this parameter(s); there was also a decrease in the expression of the latter two mutants in mitochondria. A need for a polar residue at position 34 of the hMTPPT was evident. Our findings with the positively-charged residues (i.e., His(82), His(137), Lys(231) and Lys(291)) predicted in the TMD showed that His(137) and Lys(291) are important for function (via a role in proper delivery of the protein to mitochondria). These investigations provide important information about the structure-function relationship of the hMTPPT. PMID:27188525

  19. Preliminary results from ASP on tests of QED to order. cap alpha. /sup 4/ in e/sup +/e/sup -/ annihilation at. sqrt. s = 29 GeV

    SciTech Connect

    Hawkins, C.A.

    1988-11-01

    Tests of QED to order ..cap alpha../sup 4/ performed with the ASP detector at PEP are presented. Measurements have been made of exclusive e/sup +/e/sup -/e/sup +/e/sup -/, e/sup +/e/sup -/..gamma gamma.. and ..gamma gamma gamma gamma.. final states with all particles above 50 milliradians with respect to the e/sup +/e/sup -/ beam line. These measurements represent a significant increase in statistics over previous measurements. All measurements agree well with theoretical predictions. 5 refs., 1 tab.

  20. Transmission test for linkage disequilibrium: The insulin gene region and insulin-dependent diabetes mellitus (IDDM)

    SciTech Connect

    Spielman, R.S.; McGinnis, R.E. ); Ewens, W.J. )

    1993-03-01

    A population association has consistently been observed between insulin-dependent diabetes mellitus (IDDM) and the class 1 alleles of the region of tandem-repeat DNA (5[prime] flanking polymorphism [5[prime]FP])adjacent to the insulin gene on chromosome 11p. This finding suggests that the insulin gene region contains a gene or genes contributing to IDDM susceptibility. However, several studies that have sought to show linkage with IDDM by testing for cosegregation in affected sib pairs have failed to find evidence for linkage. As means for identifying genes for complex diseases, both the association and the affected-sib-pairs approaches have limitations. It is well known that population association between a disease and a genetic marker can arise as an artifact of population structure, even in the absence of linkage. On the other hand, linkage studies with modest numbers of affected sib pairs may fail to detect linkage, especially if there is linkage heterogeneity. The authors consider an alternative method to test for linkage with a genetic marker when population association has been found. Using data from families with at least one affected child, they evaluate the transmission of the associated marker allele from a heterozygous parent to an affected offspring. This approach has been used by several investigators, but the statistical properties of the method as a test for linkage have not been investigated. In the present paper they describe the statistical basis for this transmission test for linkage disequilibrium (transmission/disequilibrium test [TDT]). They then show the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs. The TDT provides strong evidence for linkage between the 5[prime]FP and susceptibility to IDDM. 27 refs., 6 tabs.

  1. Factors Affecting Internal Blast

    NASA Astrophysics Data System (ADS)

    Granholm, R. H.; Sandusky, H. W.; Felts, J. E.

    2007-12-01

    Internal blast refers to explosion effects in confined spaces, which are dominated by the heat output of the explosive. Theoretical temperatures and pressures may not be reached due to heat losses and incomplete gas mixing. Gas mixing can have the largest effect, potentially reducing peak quasi-static pressure by a factor of two due to lack of thermal equilibrium between products and atmosphere in the space, separate from the effect of incomplete combustion of excess fuel when that atmosphere is air. Chamber and test geometry affect gas mixing, which has been inferred through temperature and pressure measurements and compared to calculations. Late-time combustion is observed for TNT compared to HMX.

  2. Seasonal affective disorder.

    PubMed

    Kurlansik, Stuart L; Ibay, Annamarie D

    2012-12-01

    Seasonal affective disorder is a combination of biologic and mood disturbances with a seasonal pattern, typically occurring in the autumn and winter with remission in the spring or summer. In a given year, about 5 percent of the U.S. population experiences seasonal affective disorder, with symptoms present for about 40 percent of the year. Although the condition is seasonally limited, patients may have significant impairment from the associated depressive symptoms. Treatment can improve these symptoms and also may be used as prophylaxis before the subsequent autumn and winter seasons. Light therapy is generally well tolerated, with most patients experiencing clinical improvement within one to two weeks after the start of treatment. To avoid relapse, light therapy should continue through the end of the winter season until spontaneous remission of symptoms in the spring or summer. Pharmacotherapy with antidepressants and cognitive behavior therapy are also appropriate treatment options and have been shown to be as effective as light therapy. Because of the comparable effectiveness of treatment options, first-line management should be guided by patient preference.

  3. Schizophrenia: A genome scan targets chromosomes 3p and 8p as potential sites of susceptibility genes

    SciTech Connect

    Pulver, A.E.; Lasseter, V.K.; Kasch, L.

    1995-06-19

    Using a systematically ascertained sample of 57 families, each having 2 or more members with a consensus diagnosis of schizophrenia (DSM-III-R criteria), we have carried out linkage studies of 520 loci, covering approximately 70% of the genome for susceptibility loci for schizophrenia. A two-stage strategy based on lod score thresholds from simulation studies of our sample identified regions for further exploration. In each region, a dense map of highly informative dinucleotide repeat polymorphisms (heterozygosity greater than .70) was analyzed using dominant, recessive, and {open_quotes}affected only{close_quotes} models and nonparametric sib pair identity-by-descent methods. For one region, 8p22-p21, affected sib-pair analyses gave a P value = .0001, corresponding to a lod score approximately equal to 3.00. For 8p22-p21, the maximum two-point lod score occurred using the {open_quotes}affected only{close_quotes} recessive model (Z{sub max} = 2.35; {theta}{sub M} = {theta}{sub F}); allowing for a constant sex difference in recombination fractions found in reference pedigrees, Z{sub max} = 2.78 ({theta}{sub M}/{theta}{sub F} = 3). For a second region, 3p26-p24, the maximum two-point lod score was 2.34 ({open_quotes}affected only{close_quotes} dominant model), and the affected sib-pair P value was .01. These two regions are worthy of further exploration as potential sites of susceptibility genes for schizophrenia. 59 refs., 2 figs., 4 tabs.

  4. Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation.

    PubMed

    Asselta, Rosanna; Robusto, Michela; Platé, Manuela; Santoro, Cristina; Peyvandi, Flora; Duga, Stefano

    2015-07-01

    Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes. In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene.

  5. Cholesterol and Affective Morbidity

    PubMed Central

    Fiedorowicz, Jess G.; Palagummi, Narasimha M.; Behrendtsen, Ole; Coryell, William H.

    2009-01-01

    Depression and mania have been linked with low cholesterol though there has been limited prospective study of cholesterol and subsequent course of affective illness. We studied the relationship between fasting total cholesterol and subsequent depressive and manic symptoms. A total of 131 participants from a prospective cohort study were identified as having had a fasting total cholesterol evaluation at intake. Participants were predominantly inpatients at index visit and were followed for a median of 20 and up to 25 years. Cholesterol was modeled with age, gender, and index use of a mood stabilizer in linear regression to assess its influence on subsequent depressive symptom burden in participants with unipolar disorder as well as depressive and manic symptom burden in participants with bipolar disorder. Among bipolar participants (N=65), low cholesterol predicted a higher proportion of follow-up weeks with manic, but not depressive symptoms. Cholesterol did not appear to predict depressive symptom burden among participants with unipolar depression (N=66). Lower cholesterol levels may predispose individuals with bipolar disorder to a greater burden of manic symptomatology and may provide some insight into the underlying neurobiology. PMID:19969372

  6. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology.

  7. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology. PMID:27535258

  8. Pteridines and affective disorders.

    PubMed

    Hoekstra, R; Fekkes, D

    2002-06-01

    The pteridine tetrahydrobiopterin (BH4) is an essential cofactor in the biosynthesis of dopamine, (nor)epinephrine, serotonin and nitric oxide (NO). Furthermore, BH4 has a direct influence on release mechanisms of these neurotransmitters and on serotonin receptor binding activity immunology. The synthesis of BH4 is stimulated by interferon-gamma and hence there is a close relationship with the immune system HPA-axis. In animal experiments it was also found that the hypothalamus-pituitary-adrenal axis influences the pteridine metabolism. In clinical studies, so far, no evidence has been found for this relationship diseases. A congenital biopterin deficiency results in atypical phenylketonuria with severe neuropsychiatric symptoms. In several neurological diseases, such as Parkinson's disease, decreased levels of BH4 are found depression. Since 1984 there have been reports on decreased biopterin and increased neopterin levels in urine and plasma of depressed patients. Conflicting results have also been found, however, due probably to methodological problems therapy. Until now, oral administration of BH4 to depressed patients has been performed by two investigators, which resulted in mainly temporal clinical improvement discussion. Understanding of biochemical mechanisms in which pteridines are involved may contribute to our knowledge of the pathogenesis and treatment of affective disorders. This paper aims to provide an overview of the relevant literature and warrant for further research on this intriguing compound. PMID:26984153

  9. Community structure affects behavior.

    PubMed

    Jaenson, C

    1991-06-01

    AID's prevention efforts can benefit from taking into account 5 main aspects (KEPRA) of community structure identified by anthropologists: 1) kinship patterns, 2) economics, 3) politics, 4) religion, and 5) associations. For example, in Uganda among the Basoga and paternal aunt or senga is responsible for female sex education. Such culturally determined patterns need to be targeted in order to enhance education and effectiveness. Economics can reflect differing systems of family support through sexual means. The example given involves a poor family with a teenager in Thailand who exchanges a water buffalo or basic necessity for this daughter's prostitution. Politics must be considered because every society identifies people who have the power to persuade, influence, exchange resources, coerce, or in some way get people to do what is wanted. Utilizing these resources whether its ministers of health, factory owners, or peers is exemplified in the Monterey, Mexico factor floor supervisor and canteen worker introducing to workers the hows and whys of a new AID's education program. His peer status will command more respect than the director with direct authority. Religious beliefs have explanations for causes of sickness or disease, or provide instruction in sex practices. The example given is of a health workers in Uganda discussing AIDS with rural women by saying that we all know that disease and deaths are caused by spells. "But not AIDS - slim. AIDS is different." Associations can help provide educational, economic, and emotional assistance to the AID's effort or families affected.

  10. Affective Incoherence: When Affective Concepts and Embodied Reactions Clash

    PubMed Central

    Centerbar, David B.; Clore, Gerald L.; Schnall, Simone; Garvin, Erika

    2008-01-01

    In five studies, we examined the effects on cognitive performance of coherence and incoherence between conceptual and experiential sources of affective information. The studies crossed the priming of happy and sad concepts with affective experiences. In different experiments, these included: approach or avoidance actions, happy or sad feelings, and happy or sad expressive behaviors. In all studies, coherence between affective concepts and affective experiences led to better recall of a story than affective incoherence. We suggested that the experience of such experiential affective cues serves as evidence of the appropriateness of affective concepts that come to mind. The results suggest that affective coherence has epistemic benefits, and that incoherence is costly, for cognitive performance. PMID:18361672

  11. Affective incoherence: when affective concepts and embodied reactions clash.

    PubMed

    Centerbar, David B; Schnall, Simone; Clore, Gerald L; Garvin, Erika D

    2008-04-01

    In five studies, the authors examined the effects on cognitive performance of coherence and incoherence between conceptual and experiential sources of affective information. The studies crossed the priming of happy and sad concepts with affective experiences. In different experiments, these included approach or avoidance actions, happy or sad feelings, and happy or sad expressive behaviors. In all studies, coherence between affective concepts and affective experiences led to better recall of a story than did affective incoherence. The authors suggest that the experience of such experiential affective cues serves as evidence of the appropriateness of affective concepts that come to mind. The results suggest that affective coherence has epistemic benefits and that incoherence is costly in terms of cognitive performance.

  12. High-resolution linkage mapping for susceptibility genes in human polygenic disease: Insulin-dependent diabetes mellitus and chromosome 11q

    PubMed Central

    Hyer, R. N.; Julier, C.; Buckley, J. D.; Trucco, M.; Rotter, J.; Spielman, R.; Barnett, A.; Bain, S.; Boitard, C.; Deschamps, I.; Todd, J. A.; Bell, J. I.; Lathrop, G. M.

    1991-01-01

    Insulin-dependent diabetes mellitus (IDDM) has a complex pattern of genetic inheritance. In addition to genes mapping to the major histocompatibility complex (MHC), several lines of evidence point to the existence of other genetic susceptibility factors. Recent studies of the nonobese diabetic mouse (NOD) model of IDDM have suggested the presence, on mouse chromosome 9, of a susceptibility gene linked to the locus encoding the T-cell antigen, Thy-1. A region on human chromosome 11q is syntenic to this region on mouse chromosome 9. We have used a set of polymorphic DNA markers from chromosome 11q to investigate this region for linkage to a susceptibility gene in 81 multiplex diabetic pedigrees. The data were investigated by maximization of lod scores over genetic models and by multiple-locus affected-sib-pair analysis. We were able to exclude the presence of a susceptibility gene (location scores < −2) throughout >90% of the chromosome 11q homology region, under the assumption that the susceptibility factor would cause >50% of affected sib pairs to share two alleles identical by descent. Theoretical estimates of the power to map susceptibility genes with a high-resolution map of linked markers in a candidate region were made, using HLA as a model locus. This result illustrates the feasibility that IDDM linkage studies using mapped sets of polymorphic DNA markers have, both for other areas of the genome in IDDM and for other polygenic diseases. The analytic approaches introduced here will be useful for affected-sib-pair studies of other complex phenotypes. PMID:1990836

  13. Encountering Science Education's Capacity to Affect and Be Affected

    ERIC Educational Resources Information Center

    Alsop, Steve

    2016-01-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science…

  14. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  15. Individual Difference Variables, Affective Differentiation, and the Structures of Affect

    PubMed Central

    Terracciano, Antonio; McCrae, Robert R.; Hagemann, Dirk; Costa, Paul T.

    2008-01-01

    Methodological arguments are usually invoked to explain variations in the structure of affect. Using self-rated affect from Italian samples (N = 600), we show that individual difference variables related to affective differentiation can moderate the observed structure. Indices of circumplexity (Browne, 1992) and congruence coefficients to the hypothesized target were used to quantify the observed structures. Results did not support the circumplex model as a universal structure. A circular structure with axes of activation and valence was approximated only among more affectively differentiated groups: students and respondents with high scores on Openness to Feelings and measures of negative emotionality. A different structure, with unipolar Positive Affect and Negative Affect factors, was observed among adults and respondents with low Openness to Feelings and negative emotionality. The observed structure of affect will depend in part on the nature of the sample studied. PMID:12932207

  16. Affective Productions of Mathematical Experience

    ERIC Educational Resources Information Center

    Walshaw, Margaret; Brown, Tony

    2012-01-01

    In underscoring the affective elements of mathematics experience, we work with contemporary readings of the work of Spinoza on the politics of affect, to understand what is included in the cognitive repertoire of the Subject. We draw on those resources to tell a pedagogical tale about the relation between cognition and affect in settings of…

  17. Affective Induction and Creative Thinking

    ERIC Educational Resources Information Center

    Fernández-Abascal, Enrique G.; Díaz, María D. Martín

    2013-01-01

    Three studies explored the relation between affect and production of creative divergent thinking, assessed with the Torrance Tests of Creative Thinking (Figural TTCT). In the first study, general, positive, and negative affect, assessed with the Positive and Negative Affect Scale (PANAS) were compared with creative production. In the second study,…

  18. Search for a gene predisposing to manic-depression on chromosome 21

    SciTech Connect

    Byerley, W.; Holik, J.; Hoff, M.; Coon, H.

    1995-06-19

    Six kindreds containing multiple cases of manic-depressive illness (MDI) were genotyped with seven highly polymorphic microsatellite loci used in the construction of an index map for chromosome 21. The kindreds were also genotyped with a microsatellite polymorphism for PFKL, a chromosome 21 locus that has shown suggestive linkage to MDI in one pedigree. Evidence of linkage was not found assuming either autosomal dominant or recessive inheritance. The nonparametric affected sib pair test did not yield significant evidence of linkage. 11 refs., 1 fig., 3 tabs.

  19. Robinow syndrome: phenotypic variability in a family with a novel intragenic ROR2 mutation.

    PubMed

    Brunetti-Pierri, Nicola; Del Gaudio, Daniela; Peters, Hartmut; Justino, Henri; Ott, Claus-Eric; Mundlos, Stefan; Bacino, Carlos A

    2008-11-01

    Robinow syndrome comprises dysmorphic facial features, short stature, brachymesomelia, segmental spine defects, and genital hypoplasia. The range of severity in this disorder is broad. We report on the clinical and molecular findings of two sib pairs from the same extended family with Robinow syndrome due to a novel intragenic ROR2 deletion involving exons 6 and 7 that could not be detected by sequencing. The affected individuals exhibited variability with respect to the cleft lip, cleft palate, and cardiac findings and for the presence in one of the patients of syringomyelia, which has not been previously reported in Robinow syndrome.

  20. Synthesis, Crystal Structure, and Magnetic Properties of a Chiral Cyanide-Bridged Bimetallic Framework K3[Mn(II)(L-asp)]6[Cr(III)(CN)6]·2H2O.

    PubMed

    Li, Li; Nishihara, Sadafumi; Inoue, Katsuya; Kurmoo, Mohamedally

    2016-01-01

    All five coordinating atoms of the amino-acid dianion L-aspartate (L-asp = NH2CH(COO)CH2COO(2-)) are found to be involved in coordination with Mn(II) in the presence of [Cr(III)(CN)6](3-) to self-assemble into a chiral three-dimensional cyanide-bridged K3[Mn(L-asp)]6[Cr(CN)6]·2H2O containing the highest ratio of Mn:Cr of 6:1. It adopts the chiral P3 (no. 143) space group consisting of zigzag Mn-OCO-Mn chains sharing edges of hexagonal channels with central [Cr(CN)6](3-), while K(+) and H2O occupy another parallel star-shaped channel. Its magnetic susceptibility above 100 K is dominated by the nearest neighbor (Mn-Cr at 5.08 and 5.31 Å) antiferromagnetic (AF) exchange interactions (θ = -15(1) K) and below 40 K by further AF interaction between Mn and Mn at 5.32 Å. It finally reaches a steady value at 4.5 K, where a bifurcation of the zero-field-cooled and field-cooled magnetizations is observed in small fields (<1 kOe). The isothermal magnetization is linear in field and deviating toward saturation above 60 kOe at 2 K. No imaginary component of the ac susceptibilities is observed. This behavior is associated with long-range antiferromagnetic order of a helical or conic nature where the magnetic sublattices are numerous [2n × (6Mn + 1Cr)], leading to a domain of sufficient size to allow for the presence of the bifurcation. A model is proposed based on the local anisotropy and symmetry multiplicity of the space group. PMID:26671258

  1. Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster.

    PubMed

    Jung, Y S; Bonagura, C A; Tilley, G J; Gao-Sheridan, H S; Armstrong, F A; Stout, C D; Burgess, B K

    2000-11-24

    All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

  2. Synthesis, Crystal Structure, and Magnetic Properties of a Chiral Cyanide-Bridged Bimetallic Framework K3[Mn(II)(L-asp)]6[Cr(III)(CN)6]·2H2O.

    PubMed

    Li, Li; Nishihara, Sadafumi; Inoue, Katsuya; Kurmoo, Mohamedally

    2016-01-01

    All five coordinating atoms of the amino-acid dianion L-aspartate (L-asp = NH2CH(COO)CH2COO(2-)) are found to be involved in coordination with Mn(II) in the presence of [Cr(III)(CN)6](3-) to self-assemble into a chiral three-dimensional cyanide-bridged K3[Mn(L-asp)]6[Cr(CN)6]·2H2O containing the highest ratio of Mn:Cr of 6:1. It adopts the chiral P3 (no. 143) space group consisting of zigzag Mn-OCO-Mn chains sharing edges of hexagonal channels with central [Cr(CN)6](3-), while K(+) and H2O occupy another parallel star-shaped channel. Its magnetic susceptibility above 100 K is dominated by the nearest neighbor (Mn-Cr at 5.08 and 5.31 Å) antiferromagnetic (AF) exchange interactions (θ = -15(1) K) and below 40 K by further AF interaction between Mn and Mn at 5.32 Å. It finally reaches a steady value at 4.5 K, where a bifurcation of the zero-field-cooled and field-cooled magnetizations is observed in small fields (<1 kOe). The isothermal magnetization is linear in field and deviating toward saturation above 60 kOe at 2 K. No imaginary component of the ac susceptibilities is observed. This behavior is associated with long-range antiferromagnetic order of a helical or conic nature where the magnetic sublattices are numerous [2n × (6Mn + 1Cr)], leading to a domain of sufficient size to allow for the presence of the bifurcation. A model is proposed based on the local anisotropy and symmetry multiplicity of the space group.

  3. Encountering science education's capacity to affect and be affected

    NASA Astrophysics Data System (ADS)

    Alsop, Steve

    2016-09-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science education. These discussions are framed by desires to transcend traditional epistemic boundaries and practices. The article concludes offering some associated ambiguities and tensions involved.

  4. Encountering science education's capacity to affect and be affected

    NASA Astrophysics Data System (ADS)

    Alsop, Steve

    2015-12-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science education. These discussions are framed by desires to transcend traditional epistemic boundaries and practices. The article concludes offering some associated ambiguities and tensions involved.

  5. Testing the Grandchildren's Received Affection Scale using Affection Exchange Theory.

    PubMed

    Mansson, Daniel H

    2013-04-01

    The purpose of this study was to test the Grandchildren's Received Affection Scale (GRAS) using Affection Exchange Theory (Floyd, 2006). In accordance with Affection Exchange Theory, it was hypothesized that grandchildren's scores on the Trait Affection Received Scale (i.e., the extent to which individuals by nature receive affection) would be related significantly and positively to their reports of received affection from their grandparents (i.e., their scores on the GRAS). Additionally, a research question was asked to explore if grandchildren's received affection from their grandparents is dependent on their grandparent's biological sex or lineage (i.e., maternal vs paternal). Thus, young adult grandchildren (N = 422) completed the GRAS and the Trait Affection Received Scale. The results of zero-order Pearson correlational analyses provided support for the hypothesis, whereas the results of MANOVAs tests only partially support extant grandparent-grandchild theory and research. These findings broaden the scope of Affection Exchange Theory and also bolster the GRAS's utility in future grandparent-grandchild affectionate communication research. PMID:23833883

  6. Testing the Grandchildren's Received Affection Scale using Affection Exchange Theory.

    PubMed

    Mansson, Daniel H

    2013-04-01

    The purpose of this study was to test the Grandchildren's Received Affection Scale (GRAS) using Affection Exchange Theory (Floyd, 2006). In accordance with Affection Exchange Theory, it was hypothesized that grandchildren's scores on the Trait Affection Received Scale (i.e., the extent to which individuals by nature receive affection) would be related significantly and positively to their reports of received affection from their grandparents (i.e., their scores on the GRAS). Additionally, a research question was asked to explore if grandchildren's received affection from their grandparents is dependent on their grandparent's biological sex or lineage (i.e., maternal vs paternal). Thus, young adult grandchildren (N = 422) completed the GRAS and the Trait Affection Received Scale. The results of zero-order Pearson correlational analyses provided support for the hypothesis, whereas the results of MANOVAs tests only partially support extant grandparent-grandchild theory and research. These findings broaden the scope of Affection Exchange Theory and also bolster the GRAS's utility in future grandparent-grandchild affectionate communication research.

  7. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-01-01

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081

  8. Diversity of the Genes Implicated in Algerian Patients Affected by Usher Syndrome.

    PubMed

    Abdi, Samia; Bahloul, Amel; Behlouli, Asma; Hardelin, Jean-Pierre; Makrelouf, Mohamed; Boudjelida, Kamel; Louha, Malek; Cheknene, Ahmed; Belouni, Rachid; Rous, Yahia; Merad, Zahida; Selmane, Djamel; Hasbelaoui, Mokhtar; Bonnet, Crystel; Zenati, Akila; Petit, Christine

    2016-01-01

    Usher syndrome (USH) is an autosomal recessive disorder characterized by a dual sensory impairment affecting hearing and vision. USH is clinically and genetically heterogeneous. Ten different causal genes have been reported. We studied the molecular bases of the disease in 18 unrelated Algerian patients by targeted-exome sequencing, and identified the causal biallelic mutations in all of them: 16 patients carried the mutations at the homozygous state and 2 at the compound heterozygous state. Nine of the 17 different mutations detected in MYO7A (1 of 5 mutations), CDH23 (4 of 7 mutations), PCDH15 (1 mutation), USH1C (1 mutation), USH1G (1 mutation), and USH2A (1 of 2 mutations), had not been previously reported. The deleterious consequences of a missense mutation of CDH23 (p.Asp1501Asn) and the in-frame single codon deletion in USH1G (p.Ala397del) on the corresponding proteins were predicted from the solved 3D-structures of extracellular cadherin (EC) domains of cadherin-23 and the sterile alpha motif (SAM) domain of USH1G/sans, respectively. In addition, we were able to show that the USH1G mutation is likely to affect the binding interface between the SAM domain and USH1C/harmonin. This should spur the use of 3D-structures, not only of isolated protein domains, but also of protein-protein interaction interfaces, to predict the functional impact of mutations detected in the USH genes.

  9. DHHC2 Affects Palmitoylation, Stability, and Functions of Tetraspanins CD9 and CD151

    PubMed Central

    Sharma, Chandan; Yang, Xiuwei H.

    2008-01-01

    Although palmitoylation markedly affects tetraspanin protein biochemistry and functions, relevant palmitoylating enzymes were not known. There are 23 mammalian “DHHC” (Asp-His-His-Cys) proteins, which presumably palmitoylate different sets of protein substrates. Among DHHC proteins tested, DHHC2 best stimulated palmitoylation of tetraspanins CD9 and CD151, whereas inactive DHHC2 (containing DH→AA or C→S mutations within the DHHC motif) failed to promote palmitoylation. Furthermore, DHHC2 associated with CD9 and CD151, but not other cell surface proteins, and DHHC2 knockdown diminished CD9 and CD151 palmitoylation. Knockdown of six other Golgi-resident DHHC proteins (DHHC3, -4, -8, -17, -18, and -21) had no effect on CD9 or CD151. DHHC2 selectively affected tetraspanin palmitoylation, but not the palmitoylations of integrin β4 subunit and bulk proteins visible in [3H]palmitate-labeled whole cell lysates. DHHC2-dependent palmitoylation also had multiple functional effects. First, it promoted physical associations between CD9 and CD151, and between α3 integrin and other proteins. Second, it protected CD151 and CD9 from lysosomal degradation. Third, the presence of DHHC2, but not other DHHC proteins, shifted cells away from a dispersed state and toward increased cell–cell contacts. PMID:18508921

  10. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis: variable phenotypic expression in three affected sisters from Mexican ancestry.

    PubMed

    Arteaga, María E; Hunziker, Walter; Teo, Audrey S M; Hillmer, Axel M; Mutchinick, Osvaldo M

    2015-02-01

    Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is a rare autosomal recessive renal disease caused by mutations in genes for the tight junction transmembrane proteins Claudin-16 (CLDN16) and Claudin-19 (CLDN19). We present the first case report of a Mexican family with three affected sisters carrying a p.Gly20Asp mutation in CLDN19 whose heterozygous mother showed evident hypercalciuria and normal low magnesemia without any other clinical, laboratory, and radiological symptoms of renal disease making of her an unsuitable donor. The affected sisters showed variable phenotypic expression including age of first symptoms, renal urinary tract infections, nephrolithiasis, nephrocalcinosis, and eye symptoms consisting in retinochoroiditis, strabismus, macular scars, bilateral anisocoria, and severe myopia and astigmatism. End stage renal disease due to renal failure needed kidney transplantation in the three of them. Interesting findings were a heterozygous mother with asymptomatic hypercalciuria warning on the need of carefully explore clinical, laboratory, kidney ultrasonograpy, and mutation status in first degree asymptomatic relatives to avoid inappropriate kidney donors; an evident variable phenotypic expression among patients; the identification of a mutation almost confined to Spanish cases and a 3.5 Mb block of genomic homozygosis strongly suggesting a common remote parental ancestor for the gene mutation reported.

  11. Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper).

    PubMed

    Polgár, J; Magnenat, E M; Peitsch, M C; Wells, T N; Clemetson, K J

    1996-11-01

    Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-M(r) group I-III phospholipases A2 (PLA2s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA2s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA2. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca(2+)-binding site of PLA2s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA2. The Ca(2+)-dependencies of the enzymic activities of ecarpholin S and rPLA2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca(2+)-binding site of Ser-49 PLA2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca(2+)-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca2+ binding. Sequence comparisons of ecarpholin S and other low-M(r) PLA2s predicts the presence of a Ser-49 group in the protein family of low-M(r) PLA2s that is distinct from the Asp-49 and Lys-49 groups. PMID:8921006

  12. [Development of the affect system].

    PubMed

    Moser, U; Von Zeppelin, I

    1996-01-01

    The authors show that the development of the affect system commences with affects of an exclusively communicative nature. These regulate the relationship between subject and object. On a different plane they also provide information on the feeling of self deriving from the interaction. Affect is seen throughout as a special kind of information. One section of the article is given over to intensity regulation and early affect defenses. The development of cognitive processes leads to the integration of affect systems and cognitive structures. In the pre-conceptual concretistic phase, fantasies change the object relation in such a way as to make unpleasant affects disappear. Only at a later stage do fantasies acquire the capacity to deal with affects. Ultimately, the affect system is grounded on an invariant relationship feeling. On a variety of different levels it displays the features typical of situation theory and the theory of the representational world, thus making it possible to entertain complex object relations. In this process the various planes of the affect system are retained and practised. Finally, the authors discuss the consequences of their remarks for the understanding of psychic disturbances and the therapies brought to bear on them. PMID:8584745

  13. Phentermine, sibutramine and affective disorders.

    PubMed

    An, Hoyoung; Sohn, Hyunjoo; Chung, Seockhoon

    2013-04-01

    A safe and effective way to control weight in patients with affective disorders is needed, and phentermine is a possible candidate. We performed a PubMed search of articles pertaining to phentermine, sibutramine, and affective disorders. We compared the studies of phentermine with those of sibutramine. The search yielded a small number of reports. Reports concerning phentermine and affective disorders reported that i) its potency in the central nervous system may be comparatively low, and ii) it may induce depression in some patients. We were unable to find more studies on the subject; thus, it is unclear presently whether phentermine use is safe in affective disorder patients. Reports regarding the association of sibutramine and affective disorders were slightly more abundant. A recent study that suggested that sibutramine may have deleterious effects in patients with a psychiatric history may provide a clue for future phentermine research. Three explanations are possible concerning the association between phentermine and affective disorders: i) phentermine, like sibutramine, may have a depression-inducing effect that affects a specific subgroup of patients, ii) phentermine may have a dose-dependent depression-inducing effect, or iii) phentermine may simply not be associated with depression. Large-scale studies with affective disorder patients focusing on these questions are needed to clarify this matter before investigation of its efficacy may be carried out and it can be used in patients with affective disorders. PMID:23678348

  14. The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

    PubMed Central

    Fotheringham, I G; Dacey, S A; Taylor, P P; Smith, T J; Hunter, M G; Finlay, M E; Primrose, S B; Parker, D M; Edwards, R M

    1986-01-01

    In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage. PMID:3521591

  15. Affect and Self-Regulation

    ERIC Educational Resources Information Center

    Malmivuori, Marja-Liisa

    2006-01-01

    This paper presents affect as an essential aspect of students' self-reflection and self-regulation. The introduced concepts of self-system and self-system process stress the importance of self-appraisals of personal competence and agency in affective responses and self-regulation in problem solving. Students are viewed as agents who constantly…

  16. Factors Affecting Willingness to Mentor

    ERIC Educational Resources Information Center

    Ghislieri, Chiara; Gatti, Paola; Quaglino, Gian Piero

    2009-01-01

    The paper presents a survey among 300 employees in Northern Italy to assess the willingness to mentor and identify the factors that affect it. Men and respondents with previous mentoring experience indicate a higher willingness to be a mentor. Willingness is affected by personal characteristics that are perceived as necessary for a mentor and the…

  17. Affect and Graphing Calculator Use

    ERIC Educational Resources Information Center

    McCulloch, Allison W.

    2011-01-01

    This article reports on a qualitative study of six high school calculus students designed to build an understanding about the affect associated with graphing calculator use in independent situations. DeBellis and Goldin's (2006) framework for affect as a representational system was used as a lens through which to understand the ways in which…

  18. Intuition, Affect, and Peculiar Beliefs

    PubMed Central

    Boden, Matthew Tyler; Berenbaum, Howard; Topper, Maurice

    2012-01-01

    Research with college students has found that intuitive thinking (e.g., using hunches to ascribe meaning to experiences) and positive affect interactively predict ideas of reference and odd/magical beliefs. We investigated whether these results would generalize to a diverse community sample of adults that included individuals with elevated levels of peculiar perceptions and beliefs. We measured positive and negative affect and intuitive thinking through questionnaires, and peculiar beliefs (i.e., ideas of reference and odd/magical beliefs) through structured clinical interviews. We found that peculiar beliefs were associated with intuitive thinking and negative affect, but not positive affect. Furthermore, in no instance did the interaction of affect and intuitive thinking predict peculiar beliefs. These results suggest that there are important differences in the factors that contribute to peculiar beliefs between college students and clinically meaningful samples. PMID:22707815

  19. Flow, affect and visual creativity.

    PubMed

    Cseh, Genevieve M; Phillips, Louise H; Pearson, David G

    2015-01-01

    Flow (being in the zone) is purported to have positive consequences in terms of affect and performance; however, there is no empirical evidence about these links in visual creativity. Positive affect often--but inconsistently--facilitates creativity, and both may be linked to experiencing flow. This study aimed to determine relationships between these variables within visual creativity. Participants performed the creative mental synthesis task to simulate the creative process. Affect change (pre- vs. post-task) and flow were measured via questionnaires. The creativity of synthesis drawings was rated objectively and subjectively by judges. Findings empirically demonstrate that flow is related to affect improvement during visual creativity. Affect change was linked to productivity and self-rated creativity, but no other objective or subjective performance measures. Flow was unrelated to all external performance measures but was highly correlated with self-rated creativity; flow may therefore motivate perseverance towards eventual excellence rather than provide direct cognitive enhancement.

  20. Daily affect and daily beliefs.

    PubMed

    Harris, Claire; Daniels, Kevin

    2005-10-01

    Human resource directorate employees of a large United Kingdom public hospital (N=36) completed an initial questionnaire and then participated in a daily diary study. The questionnaire included measures of affect and beliefs about high work demands' influence on affect and work performance. The diary included measures of affect, extent of high work demands, and daily beliefs, corresponding to those measured in the questionnaire. Participants were required to complete the diary twice daily, before and after work over a 2-week period. Measures of affect after work were associated with beliefs concerning work demands' influence on work performance and on affect measured after work. Beliefs about work demands measured in the questionnaire were associated with subsequent daily assessments of beliefs.

  1. Does income affect fertility or does fertility affect income?

    PubMed

    Bonitsis, T H; Geithman, D T

    1987-01-01

    "This paper tests for the dynamic causal connection between real income per capita and the birth rate for a subset of developing countries. These countries are Costa Rica, El Salvador, Guatemala, Mexico, and Uruguay. Our empirical findings show that, for the historical period under review, in several countries real income per capita affected the birth rate. Virtually no evidence is found to support the hypothesis that the birth rate affected real income per capita."

  2. Versatility of heme coordination demonstrated in a fungal peroxidase. Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245-->Asn mutant at various pH values.

    PubMed

    Smulevich, G; Neri, F; Marzocchi, M P; Welinder, K G

    1996-08-13

    The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly

  3. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network

    PubMed Central

    Gallego, Carlos J.; Burt, Amber; Sundaresan, Agnes S.; Ye, Zi; Shaw, Christopher; Crosslin, David R.; Crane, Paul K.; Fullerton, S. Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A.; Kitchner, Terrie E.; Ragon, Brittany K.; Stallings, Sarah C.; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E.; Aufox, Sharon A.; Pacheco, Jennifer A.; Patel, Vaibhav; Friesema, Elisha M.; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S.; Ritchie, Marylyn; Motulsky, Arno G.; Kullo, Iftikhar J.; Larson, Eric B.; Tromp, Gerard; Brilliant, Murray H.; Bottinger, Erwin; Denny, Joshua C.; Roden, Dan M.; Williams, Marc S.; Jarvik, Gail P.

    2015-01-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data. PMID:26365338

  4. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network.

    PubMed

    Gallego, Carlos J; Burt, Amber; Sundaresan, Agnes S; Ye, Zi; Shaw, Christopher; Crosslin, David R; Crane, Paul K; Fullerton, S Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A; Kitchner, Terrie E; Ragon, Brittany K; Stallings, Sarah C; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E; Aufox, Sharon A; Pacheco, Jennifer A; Patel, Vaibhav; Friesema, Elisha M; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S; Ritchie, Marylyn; Motulsky, Arno G; Kullo, Iftikhar J; Larson, Eric B; Tromp, Gerard; Brilliant, Murray H; Bottinger, Erwin; Denny, Joshua C; Roden, Dan M; Williams, Marc S; Jarvik, Gail P

    2015-10-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data. PMID:26365338

  5. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network.

    PubMed

    Gallego, Carlos J; Burt, Amber; Sundaresan, Agnes S; Ye, Zi; Shaw, Christopher; Crosslin, David R; Crane, Paul K; Fullerton, S Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A; Kitchner, Terrie E; Ragon, Brittany K; Stallings, Sarah C; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E; Aufox, Sharon A; Pacheco, Jennifer A; Patel, Vaibhav; Friesema, Elisha M; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S; Ritchie, Marylyn; Motulsky, Arno G; Kullo, Iftikhar J; Larson, Eric B; Tromp, Gerard; Brilliant, Murray H; Bottinger, Erwin; Denny, Joshua C; Roden, Dan M; Williams, Marc S; Jarvik, Gail P

    2015-10-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data.

  6. Affective modulation of attentional switching.

    PubMed

    Heerebout, Bram T; Todorović, Ana; Smedinga, Hilde E; Phaf, R Hans

    2013-01-01

    Affective modulation of attentional switching may have developed early in evolution and may therefore have primacy over other affective influences. This behavioral study investigated the influence of affect on attentional switching between emotionally neutral stimuli, whether limited-capacity control processes are involved, and whether attentional flexibility should be distinguished from attentional broadening. Experiment 1 showed that suboptimally presented happy faces facilitated switching from an automatized response routine, whereas angry faces had the opposite effect. In Experiment 2, participants with a dominant global (i.e., broad) or local (i.e., narrow) spatial bias switched more easily to the opposite bias after suboptimal happy faces than after neutral primes but less easily after angry faces. Affective modulation of attentional switching was probably incorporated during evolution in many more complex forms of information processing.

  7. Sleep Can Affect Male Fertility

    MedlinePlus

    ... page: https://medlineplus.gov/news/fullstory_161569.html Sleep Can Affect Male Fertility Study found too little ... appears to be 7 to 8 hours of sleep a night, said study author Lauren Wise, a ...

  8. Stress factors in affective diseases.

    PubMed

    Bidzińska, E J

    1984-02-01

    An investigation carried out on 97 patients with affective disorders and on 100 healthy control subjects, revealed that acute and chronic stress factors occurred more in the group of patients with affective disorders than among healthy control over a similar time period. The frequency of stressful life situations was the same before the first affective episode in patients with unipolar and bipolar illness. The possible participation of such factors in triggering the first phase of illness is discussed. Similar factors appeared in both types of affective disorders. Significantly more frequent among patients than in the control group were: marital and family conflicts, health problems, emotional and ambitional failures, lack of success and work overload.

  9. Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

    PubMed

    Lee, Kyeong-Min; Jung, Gwon-Soo; Park, Jin-Kyu; Choi, Seong-Kyoon; Jeon, Won Bae

    2013-03-01

    Extracellular matrix (ECM) plays an important role in controlling the β-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects β-cell growth characteristics and insulin secretion, β-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, β-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), β-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

  10. On Patterns in Affective Media

    NASA Astrophysics Data System (ADS)

    ADAMATZKY, ANDREW

    In computational experiments with cellular automaton models of affective solutions, where chemical species represent happiness, anger, fear, confusion and sadness, we study phenomena of space time dynamic of emotions. We demonstrate feasibility of the affective solution paradigm in example of emotional abuse therapy. Results outlined in the present paper offer unconventional but promising technique to design, analyze and interpret spatio-temporal dynamic of mass moods in crowds.

  11. Human dopamine {beta}-hydroxylase locus and the chromosome 9q34 region in alcoholism

    SciTech Connect

    Parsian. A.; Suarez, B.K.; Hampe, C.

    1994-09-01

    Human dopamine {beta}-hydroxylase (DBH) is responsible for conversion of dopamine to norepinephrine in catecholamine neurons. Potential inhibitors of this enzyme do exist, but they are generally not effective in vivo in reducing tissue concentrations of catecholamines. The gene for DBH has been localized to 9q34 by linkage analysis and in situ hybridization. Recently there have been reports indicating a suggestive evidence of linkage between DNA markers in 9q34 region and alcoholism. In order to test for this suggestive linkage, we have genotyped a sample of 134 subjects with alcoholism, 30 alcoholic families (n=302) and 92 normal controls. The alcoholic subjects are probands of multiple incidence families. The normal controls are an epidemiologically ascertained samples of middle-aged, unrelated individuals. The two groups were matched for sex and ethnic background. The markers used in this study were dinucleotide repeats in the DBH gene, and two highly informative (CA) markers (D9S64, D9S66) flanking the DBH gene. A preliminary affected-sib-pair analysis was carried out under two diagnostic schemes. Regardless of whether `probable` alcoholics are classified as unaffected (t=0.63) or affected (t=1.50), these data do not reveal a significant excess in DBH marker sharing among affected-sib-pairs. However, the comparison of the DBH marker allele frequencies between the unrelated alcoholic panel and the unrelated normal control panel was significant at the p=0.04 level.

  12. Congenital hyperreninemic hypoaldosteronism unlinked to the aldosterone synthase (CYP11B2) gene.

    PubMed

    Kayes-Wandover, K M; Tannin, G M; Shulman, D; Peled, D; Jones, K L; Karaviti, L; White, P C

    2001-11-01

    Isolated hyperreninemic hypoaldosteronism presenting in infancy is usually caused by mutations in the CYP11B2 gene encoding aldosterone synthase. We studied five patients in four unrelated kindreds with hyperreninemic hypoaldosteronism, in whom we were unable to find such mutations. All presented in infancy with failure to thrive, hyponatremia, hyperkalemia, markedly elevated plasma renin activity, and low or inappropriately normal aldosterone levels. All had normal cortisol levels and no signs or symptoms of congenital adrenal hyperplasia. All responded to fludrocortisone treatment. There were no mutations detected in exons or splice junctions of CYP11B2. Linkage of the disorder to CYP11B2 was studied in two unrelated consanguineous patients and in an affected sib pair. The consanguineous patients were each heterozygous for at least one of three polymorphic microsatellite markers near CYP11B2, excluding linkage to CYP11B2. However, linkage of the disease to CYP11B2 could not be excluded in the affected sib pair. Genes involved in the regulation of aldosterone biosynthesis, including those encoding angiotensinogen, angiotensin-converting enzyme, and the AT1 angiotensin II receptor were similarly excluded from linkage. These results demonstrate the existence of an inherited form of hyperreninemic hypoaldosteronism distinct from aldosterone synthase deficiency. The affected gene(s) remain to be determined.

  13. 16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases.

    PubMed

    Conrad, J; Niu, L; Rudd, K; Lane, B G; Ofengand, J

    1999-06-01

    The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA- MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C. Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.

  14. Charcot-Marie-Tooth neuropathy type 2 and P0 point mutations: two novel amino acid substitutions (Asp61Gly; Tyr119Cys) and a possible "hotspot" on Thr124Met.

    PubMed

    Senderek, J; Hermanns, B; Lehmann, U; Bergmann, C; Marx, G; Kabus, C; Timmerman, V; Stoltenburg-Didinger, G; Schröder, J M

    2000-04-01

    Mutations in the gene for the major protein component of peripheral nerve myelin, myelin protein zero (MPZ, P0), cause hereditary disorders of Schwann cell myelin such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), and congenital hypomyelinating neuropathy (CHN). More recently, P0 mutations were identified in the axonal type of CMT neuropathy, CMT2, which is different from the demyelinating variants with respect to electroneurography and nerve pathology. We screened 49 patients with a clinical and histopathological diagnosis of CMT2 for mutations in the P0 gene. Three heterozygous single nucleotide changes were detected: two novel missense mutations, Asp61Gly and Tyr119Cys, and the known Thr124Met substitution, that has already been reported in several CMT patients from different European countries. Haplotype analysis for the P0 locus proved that our patients with the 124Met allele were not related to a cohort of patients with the same mutation, all of Belgian descent and all found to share a common ancestor. Our data suggest that P0 mutations account for a detectable proportion of CMT2 cases with virtually every patient harbouring a different mutation but recurrence of the Thr124Met amino acid substitution. The high frequency of this peculiar genotype in the European CMT population is presumably not only due to a founder effect but Thr124Met might constitute a mutation hotspot in the P0 gene as well. PMID:10764043

  15. A polymorphic variant of human erythrocyte carbonic anhydrase I with a widespread distribution in Australian aborigines, CAI Australia-9 (8 Asp leads to Gly): purification, properties, amino acid substitution, and possible physiological significance of the variant enzyme.

    PubMed

    Jones, G L; Shaw, D C

    1982-10-01

    Carbonic anhydrase I (EC 4.2.1.1) purified from the pooled packed red blood cells of 100 individuals typed as heterozygous for the common Australian Aboriginal carbonic anhydrase I variant CAI Australia-9 had a slightly higher specific CO2 hydratase or esterase (toward p-nitrophenyl acetate) activity than the normal component and a higher Km and Vmax using the esterase substrate. The variant enzyme was slightly more resistant to heat inactivation. The extent of inhibition of both enzymes by the specific inhibitor acetazolamide was identical, as was their immunological behavior and the lability of the active-site zinc ion. The variant enzyme was more resistant to chloride inhibition. The physiological importance of this observation is discussed in the context of a proposed adaptive advantage of the variant gene in the arid western and central regions of Australia. The amino acid substitution in the Aboriginal variant of a glycine for an aspartic acid residue has been located at residue 8 from the N terminus (i.e., 8 Asp leads to Gly), by proteolytic and partial acid hydrolyses. The possible effects of this substitution on the structure and function of the molecule are discussed. PMID:6817746

  16. [Affective disorders and eating disorders].

    PubMed

    Fakra, Eric; Belzeaux, R; Azorin, J M; Adida, M

    2014-12-01

    Epidemiologic studies show a frequent co-occurence of affective and eating disorders. The incidence of one disorder in patients suffering from the other disorder is well over the incidence in the general population. Several causes could explain this increased comorbidity. First, the iatrogenic origin is detailed. Indeed, psychotropic drugs, and particularly mood stabilizers, often lead to modification in eating behaviors, generally inducing weight gain. These drugs can increase desire for food, reduce baseline metabolism or decrease motor activity. Also, affective and eating disorders share several characteristics in semiology. These similarities can not only obscure the differential diagnosis but may also attest of conjoint pathophysiological bases in the two conditions. However, genetic and biological findings so far are too sparse to corroborate this last hypothesis. Nonetheless, it is noteworthy that comorbidity of affective and eating disorders worsens patients'prognosis and is associated with more severe forms of affective disorders characterized by an earlier age of onset in the disease, higher number of mood episodes and a higher suicidality. Lastly, psychotropic drugs used in affective disorders (lithium, antiepileptic mood stabilizers, atypical antipsychotics, antidepressants) are reviewed in order to weigh their efficacy in eating disorders. This could help establish the best therapeutic option when confronted to comorbidity.

  17. [Poststroke-bipolar affective disorder].

    PubMed

    Bengesser, S A; Wurm, W E; Lackner, N; Birner, A; Reininghaus, B; Kapfhammer, H-P; Reininghaus, E

    2013-08-01

    A few weeks after suffering from a basal ganglia infarction (globus pallidus) with left-sided hemiplegia, a 23-year-old woman exhibited for the first time a pronounced mania with self-endangerment. The use of oral contraceptives was the only determinable risk factor. During the further course, the mother also developed a depressive disorder. Thus a certain genetic predisposition for affective disorders may be relevant, although this would not explain the outbreak by itself. An association between the right-sided basal ganglia infarction and the occurrence of a bipolar affective disorder has been described in the literature. Vascular or, respectively, inflammatory risk factors in synopsis with the aetiopathogenesis of bipolar affective disorders are also discussed in depth in this case report. PMID:23939559

  18. Heterozygous mutations in HSD17B4 cause juvenile peroxisomal D-bifunctional protein deficiency

    PubMed Central

    Amor, David J.; Marsh, Ashley P.L.; Storey, Elsdon; Tankard, Rick; Gillies, Greta; Delatycki, Martin B.; Pope, Kate; Bromhead, Catherine; Leventer, Richard J.; Bahlo, Melanie

    2016-01-01

    Objective: To determine the genetic cause of slowly progressive cerebellar ataxia, sensorineural deafness, and hypergonadotropic hypogonadism in 5 patients from 3 different families. Methods: The patients comprised 2 sib pairs and 1 sporadic patient. Clinical assessment included history, physical examination, and brain MRI. Linkage analysis was performed separately on the 2 sets of sib pairs using single nucleotide polymorphism microarrays, followed by analysis of the intersection of the regions. Exome sequencing was performed on 1 affected patient with variant filtering and prioritization undertaken using these intersected regions. Results: Using a combination of sequencing technologies, we identified compound heterozygous mutations in HSD17B4 in all 5 affected patients. In all 3 families, peroxisomal D-bifunctional protein (DBP) deficiency was caused by compound heterozygosity for 1 nonsense/deletion mutation and 1 missense mutation. Conclusions: We describe 5 patients with juvenile DBP deficiency from 3 different families, bringing the total number of reported patients to 14, from 8 families. This report broadens and consolidates the phenotype associated with juvenile DBP deficiency. PMID:27790638

  19. Trends affecting hospitals' human resources.

    PubMed

    Neudeck, M M

    1985-01-01

    Hospital workers at every level--from administrators to housekeepers--will be affected by the interaction of changes already underway in the healthcare industry. Societal forces that will affect the hospital workforce include demographic change, the rise of the participatory ethic and decentralization, a growing philosophy of job entitlement, and new pressures for unionization. At the same time, the industry is faced with changing manpower requirements, cost containment, and the oversupply of physicians. This article identifies some of the likely effects of these changes on hospital human resources and suggests ways that administrators can prepare for them.

  20. Clinical Judgment and Affective Disorders.

    ERIC Educational Resources Information Center

    Strohmer, Douglas C.; And Others

    1984-01-01

    Addressed the limitations of previous work on counselor clinical judgment in a study involving 20 counselors who were asked to make a series of judgments. Results suggested the judgment processes of experienced counselors making diagnoses of affective disorders differs depending on the type of judgment. (JAC)

  1. Aesthetics, Affect, and Educational Politics

    ERIC Educational Resources Information Center

    Means, Alex

    2011-01-01

    This essay explores aesthetics, affect, and educational politics through the thought of Gilles Deleuze and Jacques Ranciere. It contextualizes and contrasts the theoretical valences of their ethical and democratic projects through their shared critique of Kant. It then puts Ranciere's notion of dissensus to work by exploring it in relation to a…

  2. Factors Affecting the Tutoring Process.

    ERIC Educational Resources Information Center

    Hartman, Hope J.

    1990-01-01

    Analyzes factors internal to the tutor and tutee (i.e., cognition, metacognition, and affect) and external to them (e.g., teacher/tutor background knowledge, educational environment, content to be learned, socioeconomic status, family background, and cultural forces) that influence the tutoring process. Suggests a theoretical framework for…

  3. Political Trends Affecting Nonmetropolitan America.

    ERIC Educational Resources Information Center

    Nachtigal, Paul M.

    There are two stories about political trends affecting nonmetropolitan America. The old story, which is the story of declining rural population and declining rural influence on public policy formation, has its roots in early deliberations about governance in this country. Jefferson's republicanism focused on direct citizen involvement in decision…

  4. Sperm function in affective illness.

    PubMed

    Amsterdam, J; Winokur, A; Levin, R

    1981-04-01

    There is evidence for functional changes in the hypothalamic-pituitary-gonadal axis of patients with affective disorders. Little is known concerning spermatogenesis or sperm function in depressed men. We systematically evaluated the sperm indices in a group of depressed males complaining of diminished libido, and a healthy control group. No differences were noted in sperm parameters between the groups.

  5. Unconscious Affective Responses to Food

    PubMed Central

    Sato, Wataru; Sawada, Reiko; Kubota, Yasutaka; Toichi, Motomi; Fushiki, Tohru

    2016-01-01

    Affective or hedonic responses to food are crucial for humans, both advantageously (e.g., enhancing survival) and disadvantageously (e.g., promoting overeating and lifestyle-related disease). Although previous psychological studies have reported evidence of unconscious cognitive and behavioral processing related to food, it remains unknown whether affective reactions to food can be triggered unconsciously and its relationship with daily eating behaviors. We investigated these issues by using the subliminal affective priming paradigm. Photographs of food or corresponding mosaic images were presented in the peripheral visual field for 33 ms. Target photos of faces with emotionally neutral expressions were then presented, and participants rated their preferences for the faces. Eating behaviors were also assessed using questionnaires. The food images, relative to the mosaics, increased participants’ preference for subsequent target faces. Furthermore, the difference in the preference induced by food versus mosaic images was positively correlated with the tendency to engage in external eating. These results suggest that unconscious affective reactions are elicited by the sight of food and that these responses contribute to daily eating behaviors related to overeating. PMID:27501443

  6. Test Expectancy Affects Metacomprehension Accuracy

    ERIC Educational Resources Information Center

    Thiede, Keith W.; Wiley, Jennifer; Griffin, Thomas D.

    2011-01-01

    Background: Theory suggests that the accuracy of metacognitive monitoring is affected by the cues used to judge learning. Researchers have improved monitoring accuracy by directing attention to more appropriate cues; however, this is the first study to more directly point students to more appropriate cues using instructions regarding tests and…

  7. Motor Execution Affects Action Prediction

    ERIC Educational Resources Information Center

    Springer, Anne; Brandstadter, Simone; Liepelt, Roman; Birngruber, Teresa; Giese, Martin; Mechsner, Franz; Prinz, Wolfgang

    2011-01-01

    Previous studies provided evidence of the claim that the prediction of occluded action involves real-time simulation. We report two experiments that aimed to study how real-time simulation is affected by simultaneous action execution under conditions of full, partial or no overlap between observed and executed actions. This overlap was analysed by…

  8. Supersonic Wave Interference Affecting Stability

    NASA Technical Reports Server (NTRS)

    Love, Eugene S.

    1958-01-01

    Some of the significant interference fields that may affect stability of aircraft at supersonic speeds are briefly summarized. Illustrations and calculations are presented to indicate the importance of interference fields created by wings, bodies, wing-body combinations, jets, and nacelles.

  9. [Faces affect recognition in schizophrenia].

    PubMed

    Prochwicz, Katarzyna; Rózycka, Jagoda

    2012-01-01

    Clinical observations and the results of many experimental researches indicate that individuals suffering from schizophrenia reveal difficulties in the recognition of emotional states experienced by other people; however the causes and the range of these problems have not been clearly described. Despite early research results confirming that difficulties in emotion recognition are related only to negative emotions, the results of the researches conducted over the lat 30 years indicate that emotion recognition problems are a manifestation of a general cognitive deficit, and they do not concern specific emotions. The article contains a review of the research on face affect recognition in schizophrenia. It discusses the causes of these difficulties, the differences in the accuracy of the recognition of specific emotions, the relationship between the symptoms of schizophrenia and the severity of problems with face perception, and the types of cognitive processes which influence the disturbances in face affect recognition. Particular attention was paid to the methodology of the research on face affect recognition, including the methods used in control tasks relying on the identification of neutral faces designed to assess the range of deficit underlying the face affect recognition problems. The analysis of methods used in particular researches revealed some weaknesses. The article also deals with the question of the possibilities of improving the ability to recognise the emotions, and briefly discusses the efficiency of emotion recognition training programs designed for patients suffering from schizophrenia.

  10. How Supplementation Affects Grazing Behavior

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Researchers are still in the early stages of understanding how supplementation affects grazing behavior. Conventional nutrition wisdom, including early research with grazing cattle, has been based almost entirely upon stored feeds fed in confinement. In these situations, most dietary “choices” were ...

  11. Affective Experience in a Classroom.

    ERIC Educational Resources Information Center

    Anderson, Ariel; And Others

    This study introduces a novel methodology for examining affective experiences in the classroom. The general procedure is to make auditory and visual recordings of a particular child during a normal classroom period. That child is then immediately taken from the class and placed in a private room where the recordings are replayed. The student is…