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Sample records for human polya site

  1. POLYAR, a new computer program for prediction of poly(A sites in human sequences

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    Qamar Raheel

    2010-11-01

    Full Text Available Abstract Background mRNA polyadenylation is an essential step of pre-mRNA processing in eukaryotes. Accurate prediction of the pre-mRNA 3'-end cleavage/polyadenylation sites is important for defining the gene boundaries and understanding gene expression mechanisms. Results 28761 human mapped poly(A sites have been classified into three classes containing different known forms of polyadenylation signal (PAS or none of them (PAS-strong, PAS-weak and PAS-less, respectively and a new computer program POLYAR for the prediction of poly(A sites of each class was developed. In comparison with polya_svm (till date the most accurate computer program for prediction of poly(A sites while searching for PAS-strong poly(A sites in human sequences, POLYAR had a significantly higher prediction sensitivity (80.8% versus 65.7% and specificity (66.4% versus 51.7% However, when a similar sort of search was conducted for PAS-weak and PAS-less poly(A sites, both programs had a very low prediction accuracy, which indicates that our knowledge about factors involved in the determination of the poly(A sites is not sufficient to identify such polyadenylation regions. Conclusions We present a new classification of polyadenylation sites into three classes and a novel computer program POLYAR for prediction of poly(A sites/regions of each of the class. In tests, POLYAR shows high accuracy of prediction of the PAS-strong poly(A sites, though this program's efficiency in searching for PAS-weak and PAS-less poly(A sites is not very high but is comparable to other available programs. These findings suggest that additional characteristics of such poly(A sites remain to be elucidated. POLYAR program with a stand-alone version for downloading is available at http://cub.comsats.edu.pk/polyapredict.htm.

  2. Prediction of Poly(A Sites by Poly(A Read Mapping.

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    Thomas Bonfert

    Full Text Available RNA-seq reads containing part of the poly(A tail of transcripts (denoted as poly(A reads provide the most direct evidence for the position of poly(A sites in the genome. However, due to reduced coverage of poly(A tails by reads, poly(A reads are not routinely identified during RNA-seq mapping. Nevertheless, recent studies for several herpesviruses successfully employed mapping of poly(A reads to identify herpesvirus poly(A sites using different strategies and customized programs. To more easily allow such analyses without requiring additional programs, we integrated poly(A read mapping and prediction of poly(A sites into our RNA-seq mapping program ContextMap 2. The implemented approach essentially generalizes previously used poly(A read mapping approaches and combines them with the context-based approach of ContextMap 2 to take into account information provided by other reads aligned to the same location. Poly(A read mapping using ContextMap 2 was evaluated on real-life data from the ENCODE project and compared against a competing approach based on transcriptome assembly (KLEAT. This showed high positive predictive value for our approach, evidenced also by the presence of poly(A signals, and considerably lower runtime than KLEAT. Although sensitivity is low for both methods, we show that this is in part due to a high extent of spurious results in the gold standard set derived from RNA-PET data. Sensitivity improves for poly(A sites of known transcripts or determined with a more specific poly(A sequencing protocol and increases with read coverage on transcript ends. Finally, we illustrate the usefulness of the approach in a high read coverage scenario by a re-analysis of published data for herpes simplex virus 1. Thus, with current trends towards increasing sequencing depth and read length, poly(A read mapping will prove to be increasingly useful and can now be performed automatically during RNA-seq mapping with ContextMap 2.

  3. Dragon polya spotter: Predictor of poly(A) motifs within human genomic DNA sequences

    KAUST Repository

    Kalkatawi, Manal M.

    2011-11-15

    Motivation: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants. The Author(s) 2011. Published by Oxford University Press. All rights reserved.

  4. Dragon polya spotter: Predictor of poly(A) motifs within human genomic DNA sequences

    KAUST Repository

    Kalkatawi, Manal M.; Rangkuti, Farania; Schramm, Michael C.; Jankovic, Boris R.; Kamau, Allan; Chowdhary, Rajesh; Archer, John A.C.; Bajic, Vladimir B.

    2011-01-01

    . These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity

  5. Poly(A)-tag deep sequencing data processing to extract poly(A) sites.

    Science.gov (United States)

    Wu, Xiaohui; Ji, Guoli; Li, Qingshun Quinn

    2015-01-01

    Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

  6. Poly(A) motif prediction using spectral latent features from human DNA sequences

    KAUST Repository

    Xie, Bo; Jankovic, Boris R.; Bajic, Vladimir B.; Song, Le; Gao, Xin

    2013-01-01

    Motivation: Polyadenylation is the addition of a poly(A) tail to an RNA molecule. Identifying DNA sequence motifs that signal the addition of poly(A) tails is essential to improved genome annotation and better understanding of the regulatory mechanisms and stability of mRNA.Existing poly(A) motif predictors demonstrate that information extracted from the surrounding nucleotide sequences of candidate poly(A) motifs can differentiate true motifs from the false ones to a great extent. A variety of sophisticated features has been explored, including sequential, structural, statistical, thermodynamic and evolutionary properties. However, most of these methods involve extensive manual feature engineering, which can be time-consuming and can require in-depth domain knowledge.Results: We propose a novel machine-learning method for poly(A) motif prediction by marrying generative learning (hidden Markov models) and discriminative learning (support vector machines). Generative learning provides a rich palette on which the uncertainty and diversity of sequence information can be handled, while discriminative learning allows the performance of the classification task to be directly optimized. Here, we used hidden Markov models for fitting the DNA sequence dynamics, and developed an efficient spectral algorithm for extracting latent variable information from these models. These spectral latent features were then fed into support vector machines to fine-tune the classification performance.We evaluated our proposed method on a comprehensive human poly(A) dataset that consists of 14 740 samples from 12 of the most abundant variants of human poly(A) motifs. Compared with one of the previous state-of-the-art methods in the literature (the random forest model with expert-crafted features), our method reduces the average error rate, false-negative rate and false-positive rate by 26, 15 and 35%, respectively. Meanwhile, our method makes ?30% fewer error predictions relative to the other

  7. Poly(A) motif prediction using spectral latent features from human DNA sequences

    KAUST Repository

    Xie, Bo

    2013-06-21

    Motivation: Polyadenylation is the addition of a poly(A) tail to an RNA molecule. Identifying DNA sequence motifs that signal the addition of poly(A) tails is essential to improved genome annotation and better understanding of the regulatory mechanisms and stability of mRNA.Existing poly(A) motif predictors demonstrate that information extracted from the surrounding nucleotide sequences of candidate poly(A) motifs can differentiate true motifs from the false ones to a great extent. A variety of sophisticated features has been explored, including sequential, structural, statistical, thermodynamic and evolutionary properties. However, most of these methods involve extensive manual feature engineering, which can be time-consuming and can require in-depth domain knowledge.Results: We propose a novel machine-learning method for poly(A) motif prediction by marrying generative learning (hidden Markov models) and discriminative learning (support vector machines). Generative learning provides a rich palette on which the uncertainty and diversity of sequence information can be handled, while discriminative learning allows the performance of the classification task to be directly optimized. Here, we used hidden Markov models for fitting the DNA sequence dynamics, and developed an efficient spectral algorithm for extracting latent variable information from these models. These spectral latent features were then fed into support vector machines to fine-tune the classification performance.We evaluated our proposed method on a comprehensive human poly(A) dataset that consists of 14 740 samples from 12 of the most abundant variants of human poly(A) motifs. Compared with one of the previous state-of-the-art methods in the literature (the random forest model with expert-crafted features), our method reduces the average error rate, false-negative rate and false-positive rate by 26, 15 and 35%, respectively. Meanwhile, our method makes ?30% fewer error predictions relative to the other

  8. PolyaPeak: Detecting Transcription Factor Binding Sites from ChIP-seq Using Peak Shape Information

    Science.gov (United States)

    Wu, Hao; Ji, Hongkai

    2014-01-01

    ChIP-seq is a powerful technology for detecting genomic regions where a protein of interest interacts with DNA. ChIP-seq data for mapping transcription factor binding sites (TFBSs) have a characteristic pattern: around each binding site, sequence reads aligned to the forward and reverse strands of the reference genome form two separate peaks shifted away from each other, and the true binding site is located in between these two peaks. While it has been shown previously that the accuracy and resolution of binding site detection can be improved by modeling the pattern, efficient methods are unavailable to fully utilize that information in TFBS detection procedure. We present PolyaPeak, a new method to improve TFBS detection by incorporating the peak shape information. PolyaPeak describes peak shapes using a flexible Pólya model. The shapes are automatically learnt from the data using Minorization-Maximization (MM) algorithm, then integrated with the read count information via a hierarchical model to distinguish true binding sites from background noises. Extensive real data analyses show that PolyaPeak is capable of robustly improving TFBS detection compared with existing methods. An R package is freely available. PMID:24608116

  9. A Simple Decision Rule for Recognition of Poly(A) Tail Signal Motifs in Human Genome

    KAUST Repository

    AbouEisha, Hassan M.

    2015-05-12

    Background is the numerous attempts were made to predict motifs in genomic sequences that correspond to poly (A) tail signals. Vast portion of this effort has been directed to a plethora of nonlinear classification methods. Even when such approaches yield good discriminant results, identifying dominant features of regulatory mechanisms nevertheless remains a challenge. In this work, we look at decision rules that may help identifying such features. Findings are we present a simple decision rule for classification of candidate poly (A) tail signal motifs in human genomic sequence obtained by evaluating features during the construction of gradient boosted trees. We found that values of a single feature based on the frequency of adenine in the genomic sequence surrounding candidate signal and the number of consecutive adenine molecules in a well-defined region immediately following the motif displays good discriminative potential in classification of poly (A) tail motifs for samples covered by the rule. Conclusions is the resulting simple rule can be used as an efficient filter in construction of more complex poly(A) tail motifs classification algorithms.

  10. Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

    Science.gov (United States)

    Kissopoulou, Antheia; Jonasson, Jon; Lindahl, Tomas L.; Osman, Abdimajid

    2013-01-01

    Background Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. Results Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. Conclusion The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and

  11. Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

    KAUST Repository

    Schlackow, M.

    2013-10-23

    Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3\\' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3\\'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

  12. Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

    KAUST Repository

    Schlackow, M.; Marguerat, S.; Proudfoot, N. J.; Bahler, J.; Erban, R.; Gullerova, M.

    2013-01-01

    Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

  13. Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

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    Antheia Kissopoulou

    Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

  14. Omni-PolyA: a method and tool for accurate recognition of Poly(A) signals in human genomic DNA

    KAUST Repository

    Magana-Mora, Arturo

    2017-08-15

    BackgroundPolyadenylation is a critical stage of RNA processing during the formation of mature mRNA, and is present in most of the known eukaryote protein-coding transcripts and many long non-coding RNAs. The correct identification of poly(A) signals (PAS) not only helps to elucidate the 3′-end genomic boundaries of a transcribed DNA region and gene regulatory mechanisms but also gives insight into the multiple transcript isoforms resulting from alternative PAS. Although progress has been made in the in-silico prediction of genomic signals, the recognition of PAS in DNA genomic sequences remains a challenge.ResultsIn this study, we analyzed human genomic DNA sequences for the 12 most common PAS variants. Our analysis has identified a set of features that helps in the recognition of true PAS, which may be involved in the regulation of the polyadenylation process. The proposed features, in combination with a recognition model, resulted in a novel method and tool, Omni-PolyA. Omni-PolyA combines several machine learning techniques such as different classifiers in a tree-like decision structure and genetic algorithms for deriving a robust classification model. We performed a comparison between results obtained by state-of-the-art methods, deep neural networks, and Omni-PolyA. Results show that Omni-PolyA significantly reduced the average classification error rate by 35.37% in the prediction of the 12 considered PAS variants relative to the state-of-the-art results.ConclusionsThe results of our study demonstrate that Omni-PolyA is currently the most accurate model for the prediction of PAS in human and can serve as a useful complement to other PAS recognition methods. Omni-PolyA is publicly available as an online tool accessible at www.cbrc.kaust.edu.sa/omnipolya/.

  15. Useful Bicistronic Reporter System for Studying Poly(A Site-Defining cis Elements and Regulation of Alternative Polyadenylation

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    Zhongyuan Deng

    2018-01-01

    Full Text Available The link between polyadenylation (pA and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES. Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

  16. Behavior of adsorbed Poly-A onto sodium montmorillonite

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    Palomino-Aquino, Nayeli [Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (Mexico); Negrón-Mendoza, Alicia, E-mail: negron@nucleares.unam.mx [Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México (Mexico)

    2015-07-23

    The adsorption of Poly-A (a polynucleotide consisting of adenine, ribose and a phosphate group), onto a clay mineral, was studied to investigate the extent of adsorption, the site of binding, and the capacity of the clay to protect Poly-A, while it is adsorbed onto the clay, from external sources of energy. The results showed that Poly-A presented a high percentage of adsorption at the edges of the clay and that the survival of the polynucleotide was superior to irradiating the polymer in the absence of the clay.

  17. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

    Science.gov (United States)

    Ray, Swagat; Anderson, Emma C

    2016-03-03

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

  18. pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A Sequences

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    Alexandra E Grier

    2016-01-01

    Full Text Available Increasing demand for large-scale synthesis of in vitro transcribed (IVT mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A tail lengths to ≃120 base pairs (bp. Here, we have developed a novel method for generation of extended poly(A tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A tracts up to ≃500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s.

  19. The 5'-poly(A leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

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    Pragyesh Dhungel

    2017-08-01

    Full Text Available The poly(A leader at the 5'-untranslated region (5'-UTR is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs. These poly(A leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A leaders are 8-12 residues, the result suggests that the poly(A leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES. These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

  20. Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase

    International Nuclear Information System (INIS)

    Rose, K.M.; Allen, M.S.; Crawford, I.L.; Jacob, S.T.; Pennsylvania State Univ., Hershey; Texas Univ., Dallas; Texas Univ., Dallas

    1978-01-01

    The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-phenanthroline on purified nuclear poly(A) polymerase. This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitor levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only zinc was present in detectable quantities, at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much zinc as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labelled in vivo by injection of tumor-bearing animals with 65 Zn. Dialysis experiments with highly purified radiolabelled poly(A) polymerase showed that the enzyme-zinc complex was labile and that a reduction in 65 Zn content correlated with a loss in enzyme activity. (orig./AJ) [de

  1. Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Rose, K M; Allen, M S; Crawford, I L; Jacob, S T [Pennsylvania State Univ., Hershey (USA). Dept. of Pharmacology; Pennsylvania State Univ., Hershey (USA). Specialized Cancer Research Center; Texas Univ., Dallas (USA). Dept. of Neurology; Texas Univ., Dallas (USA). Dept. of Pharmacology)

    1978-07-01

    The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-Phenanthroline on purified nuclear poly(A) polymerase. This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitor levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only zinc was present in detectable quantities at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much zinc as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labelled in vivo by injection of tumor-bearing animals with /sup 65/Zn. Dialysis experiments with highly purified radiolabelled poly(A) polymerase showed that the enzyme-zinc complex was labile and that a reduction in /sup 65/Zn content correlated with a loss in enzyme activity.

  2. Regression analysis using dependent Polya trees.

    Science.gov (United States)

    Schörgendorfer, Angela; Branscum, Adam J

    2013-11-30

    Many commonly used models for linear regression analysis force overly simplistic shape and scale constraints on the residual structure of data. We propose a semiparametric Bayesian model for regression analysis that produces data-driven inference by using a new type of dependent Polya tree prior to model arbitrary residual distributions that are allowed to evolve across increasing levels of an ordinal covariate (e.g., time, in repeated measurement studies). By modeling residual distributions at consecutive covariate levels or time points using separate, but dependent Polya tree priors, distributional information is pooled while allowing for broad pliability to accommodate many types of changing residual distributions. We can use the proposed dependent residual structure in a wide range of regression settings, including fixed-effects and mixed-effects linear and nonlinear models for cross-sectional, prospective, and repeated measurement data. A simulation study illustrates the flexibility of our novel semiparametric regression model to accurately capture evolving residual distributions. In an application to immune development data on immunoglobulin G antibodies in children, our new model outperforms several contemporary semiparametric regression models based on a predictive model selection criterion. Copyright © 2013 John Wiley & Sons, Ltd.

  3. On Polya's inequality for torsional rigidity and first Dirichlet eigenvalue

    OpenAIRE

    Berg, M. van den; Ferone, V.; Nitsch, C.; Trombetti, C.

    2016-01-01

    Let $\\Omega$ be an open set in Euclidean space with finite Lebesgue measure $|\\Omega|$. We obtain some properties of the set function $F:\\Omega\\mapsto \\R^+$ defined by $$ F(\\Omega)=\\frac{T(\\Omega)\\lambda_1(\\Omega)}{|\\Omega|} ,$$ where $T(\\Omega)$ and $\\lambda_1(\\Omega)$ are the torsional rigidity and the first eigenvalue of the Dirichlet Laplacian respectively. We improve the classical P\\'olya bound $F(\\Omega)\\le 1,$ and show that $$F(\\Omega)\\le 1- \

  4. LEMBAR KERJA PESERTA DIDIK (LKPD BERBASIS PROBLEM SOLVING POLYA

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    Lilis Nurliawaty

    2017-03-01

    Full Text Available Lack of exact use of teaching materials and does not correspond to the needs of student leads to lack of analytical ability of students to the process of problem solving. Research development worksheets based on Polya problem solving on the heat material aims to develop valid LKPD, practical, and effective. Stages of development using the 4D model was modified into 3D, namely define (definition, Design (planning, and Development (development The results of the validity of the learning device in the category valid, obtained from the calculation of CVI are in the range 0-1 and said in category reliably with r11 value greater than rtabel (rcount > rtabel. The results of the analysis of questionnaire responses of students obtained an average percentage of 87.9% on the analysis. The analysis result of sheets assessment of learning physics used LKPD-based Polya problem solving obtained average percentage analysis results in the first meeting is 77.33% with good category, the average percentage of the results of the analysis at the second meeting is 81.11% with a very good category and average of results percentage analysis at the third meeting is 78.89% with good category. So it can say that LKPD-based Polya problem solving developed valid, practical and effective to use.

  5. Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

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    Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

    2018-03-19

    To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

  6. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

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    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  7. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    Science.gov (United States)

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  8. Bayesian nonparametric meta-analysis using Polya tree mixture models.

    Science.gov (United States)

    Branscum, Adam J; Hanson, Timothy E

    2008-09-01

    Summary. A common goal in meta-analysis is estimation of a single effect measure using data from several studies that are each designed to address the same scientific inquiry. Because studies are typically conducted in geographically disperse locations, recent developments in the statistical analysis of meta-analytic data involve the use of random effects models that account for study-to-study variability attributable to differences in environments, demographics, genetics, and other sources that lead to heterogeneity in populations. Stemming from asymptotic theory, study-specific summary statistics are modeled according to normal distributions with means representing latent true effect measures. A parametric approach subsequently models these latent measures using a normal distribution, which is strictly a convenient modeling assumption absent of theoretical justification. To eliminate the influence of overly restrictive parametric models on inferences, we consider a broader class of random effects distributions. We develop a novel hierarchical Bayesian nonparametric Polya tree mixture (PTM) model. We present methodology for testing the PTM versus a normal random effects model. These methods provide researchers a straightforward approach for conducting a sensitivity analysis of the normality assumption for random effects. An application involving meta-analysis of epidemiologic studies designed to characterize the association between alcohol consumption and breast cancer is presented, which together with results from simulated data highlight the performance of PTMs in the presence of nonnormality of effect measures in the source population.

  9. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  10. RNA polyadenylation sites on the genomes of microorganisms, animals, and plants.

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    Xiu-Qing Li

    Full Text Available Pre-messenger RNA (mRNA 3'-end cleavage and subsequent polyadenylation strongly regulate gene expression. In comparison with the upstream or downstream motifs, relatively little is known about the feature differences of polyadenylation [poly(A] sites among major kingdoms. We suspect that the precise poly(A sites are very selective, and we therefore mapped mRNA poly(A sites on complete and nearly complete genomes using mRNA sequences available in the National Center for Biotechnology Information (NCBI Nucleotide database. In this paper, we describe the mRNA nucleotide [i.e., the poly(A tail attachment position] that is directly in attachment with the poly(A tail and the pre-mRNA nucleotide [i.e., the poly(A tail starting position] that corresponds to the first adenosine of the poly(A tail in the 29 most-mapped species (2 fungi, 2 protists, 18 animals, and 7 plants. The most representative pre-mRNA dinucleotides covering these two positions were UA, CA, and GA in 17, 10, and 2 of the species, respectively. The pre-mRNA nucleotide at the poly(A tail starting position was typically an adenosine [i.e., A-type poly(A sites], sometimes a uridine, and occasionally a cytidine or guanosine. The order was U>C>G at the attachment position but A>>U>C≥G at the starting position. However, in comparison with the mRNA nucleotide composition (base composition, the poly(A tail attachment position selected C over U in plants and both C and G over U in animals, in both A-type and non-A-type poly(A sites. Animals, dicot plants, and monocot plants had clear differences in C/G ratios at the poly(A tail attachment position of the non-A-type poly(A sites. This study of poly(A site evolution indicated that the two positions within poly(A sites had distinct nucleotide compositions and were different among kingdoms.

  11. The effects of Polya's heuristic and diary writing on children's problem solving

    Science.gov (United States)

    Hensberry, Karina K. R.; Jacobbe, Tim

    2012-03-01

    This paper presents the results of a study that aimed at increasing students' problem-solving skills. Polya's (1985) heuristic for problem solving was used and students were required to articulate their thought processes through the use of a structured diary. The diary prompted students to answer questions designed to engage them in the phases of Polya's (1985) heuristic. While it appeared as though most students did not internalise the diary questions, further analysis of students' responses indicated that most students showed improvement in their solution strategies. These results indicate that having students write about their thinking may be beneficial for developing their problem-solving skills.

  12. The Effects of Polya's Heuristic and Diary Writing on Children's Problem Solving

    Science.gov (United States)

    Hensberry, Karina K. R.; Jacobbe, Tim

    2012-01-01

    This paper presents the results of a study that aimed at increasing students' problem-solving skills. Polya's (1985) heuristic for problem solving was used and students were required to articulate their thought processes through the use of a structured diary. The diary prompted students to answer questions designed to engage them in the phases of…

  13. Students' Errors in Solving the Permutation and Combination Problems Based on Problem Solving Steps of Polya

    Science.gov (United States)

    Sukoriyanto; Nusantara, Toto; Subanji; Chandra, Tjang Daniel

    2016-01-01

    This article was written based on the results of a study evaluating students' errors in problem solving of permutation and combination in terms of problem solving steps according to Polya. Twenty-five students were asked to do four problems related to permutation and combination. The research results showed that the students still did a mistake in…

  14. An Improved Estimation Using Polya-Gamma Augmentation for Bayesian Structural Equation Models with Dichotomous Variables

    Science.gov (United States)

    Kim, Seohyun; Lu, Zhenqiu; Cohen, Allan S.

    2018-01-01

    Bayesian algorithms have been used successfully in the social and behavioral sciences to analyze dichotomous data particularly with complex structural equation models. In this study, we investigate the use of the Polya-Gamma data augmentation method with Gibbs sampling to improve estimation of structural equation models with dichotomous variables.…

  15. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  16. The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A polymerase in Entamoeba histolytica.

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    Marisol Pezet-Valdez

    Full Text Available In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25 from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25 was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A polymerase (EhPAP that is responsible for the synthesis of the poly(A tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

  17. Recurrence and Polya Number of General One-Dimensional Random Walks

    International Nuclear Information System (INIS)

    Zhang Xiaokun; Wan Jing; Lu Jingju; Xu Xinping

    2011-01-01

    The recurrence properties of random walks can be characterized by Polya number, i.e., the probability that the walker has returned to the origin at least once. In this paper, we consider recurrence properties for a general 1D random walk on a line, in which at each time step the walker can move to the left or right with probabilities l and r, or remain at the same position with probability o (l + r + o = 1). We calculate Polya number P of this model and find a simple expression for P as, P = 1 - Δ, where Δ is the absolute difference of l and r (Δ = |l - r|). We prove this rigorous expression by the method of creative telescoping, and our result suggests that the walk is recurrent if and only if the left-moving probability l equals to the right-moving probability r. (general)

  18. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  19. PATACSDB—the database of polyA translational attenuators in coding sequences

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    Malgorzata Habich

    2016-02-01

    Full Text Available Recent additions to the repertoire of gene expression regulatory mechanisms are polyadenylate (polyA tracks encoding for poly-lysine runs in protein sequences. Such tracks stall the translation apparatus and induce frameshifting independently of the effects of charged nascent poly-lysine sequence on the ribosome exit channel. As such, they substantially influence the stability of mRNA and the amount of protein produced from a given transcript. Single base changes in these regions are enough to exert a measurable response on both protein and mRNA abundance; this makes each of these sequences a potentially interesting case study for the effects of synonymous mutation, gene dosage balance and natural frameshifting. Here we present PATACSDB, a resource that contain a comprehensive list of polyA tracks from over 250 eukaryotic genomes. Our data is based on the Ensembl genomic database of coding sequences and filtered with algorithm of 12A-1 which selects sequences of polyA tracks with a minimal length of 12 A’s allowing for one mismatched base. The PATACSDB database is accessible at: http://sysbio.ibb.waw.pl/patacsdb. The source code is available at http://github.com/habich/PATACSDB, and it includes the scripts with which the database can be recreated.

  20. Site specific modification of the human plasma proteome by methylglyoxal

    International Nuclear Information System (INIS)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig S.; Monks, Terrence J.; Lau, Serrine S.

    2015-01-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  1. Site specific modification of the human plasma proteome by methylglyoxal

    Energy Technology Data Exchange (ETDEWEB)

    Kimzey, Michael J.; Kinsky, Owen R. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Yassine, Hussein N. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Tsaprailis, George [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Stump, Craig S. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Southern Arizona VA Health Care System, Tucson, AZ 85723 (United States); Monks, Terrence J. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Lau, Serrine S., E-mail: lau@pharmacy.arizona.edu [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States)

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  2. Pervasive hitchhiking at coding and regulatory sites in humans.

    Directory of Open Access Journals (Sweden)

    James J Cai

    2009-01-01

    Full Text Available Much effort and interest have focused on assessing the importance of natural selection, particularly positive natural selection, in shaping the human genome. Although scans for positive selection have identified candidate loci that may be associated with positive selection in humans, such scans do not indicate whether adaptation is frequent in general in humans. Studies based on the reasoning of the MacDonald-Kreitman test, which, in principle, can be used to evaluate the extent of positive selection, suggested that adaptation is detectable in the human genome but that it is less common than in Drosophila or Escherichia coli. Both positive and purifying natural selection at functional sites should affect levels and patterns of polymorphism at linked nonfunctional sites. Here, we search for these effects by analyzing patterns of neutral polymorphism in humans in relation to the rates of recombination, functional density, and functional divergence with chimpanzees. We find that the levels of neutral polymorphism are lower in the regions of lower recombination and in the regions of higher functional density or divergence. These correlations persist after controlling for the variation in GC content, density of simple repeats, selective constraint, mutation rate, and depth of sequencing coverage. We argue that these results are most plausibly explained by the effects of natural selection at functional sites -- either recurrent selective sweeps or background selection -- on the levels of linked neutral polymorphism. Natural selection at both coding and regulatory sites appears to affect linked neutral polymorphism, reducing neutral polymorphism by 6% genome-wide and by 11% in the gene-rich half of the human genome. These findings suggest that the effects of natural selection at linked sites cannot be ignored in the study of neutral human polymorphism.

  3. Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

    Science.gov (United States)

    Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

    Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

  4. Canonical Poly(A Polymerase Activity Promotes the Decay of a Wide Variety of Mammalian Nuclear RNAs.

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    Stefan M Bresson

    2015-10-01

    Full Text Available The human nuclear poly(A-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs. In addition, PABPN1 promotes hyperadenylation by stimulating poly(A-polymerases (PAPα/γ, but this activity has not previously been linked to the decay of endogenous transcripts. Moreover, the mechanisms underlying target specificity have remained elusive. Here, we inactivated PAP-dependent hyperadenylation in cells by two independent mechanisms and used an RNA-seq approach to identify endogenous targets. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and promoter upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD. Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. Additional investigation showed that a genetically-encoded poly(A tail is sufficient to drive decay, suggesting that degradation occurs independently of the canonical cleavage and polyadenylation reaction. Surprisingly, treatment with transcription inhibitors uncouples polyadenylation from decay, leading to runaway hyperadenylation of nuclear decay targets. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts.

  5. Human-Robot Site Survey and Sampling for Space Exploration

    Science.gov (United States)

    Fong, Terrence; Bualat, Maria; Edwards, Laurence; Flueckiger, Lorenzo; Kunz, Clayton; Lee, Susan Y.; Park, Eric; To, Vinh; Utz, Hans; Ackner, Nir

    2006-01-01

    NASA is planning to send humans and robots back to the Moon before 2020. In order for extended missions to be productive, high quality maps of lunar terrain and resources are required. Although orbital images can provide much information, many features (local topography, resources, etc) will have to be characterized directly on the surface. To address this need, we are developing a system to perform site survey and sampling. The system includes multiple robots and humans operating in a variety of team configurations, coordinated via peer-to-peer human-robot interaction. In this paper, we present our system design and describe planned field tests.

  6. Characterization of Rous sarcoma virus polyadenylation site use in vitro

    International Nuclear Information System (INIS)

    Maciolek, Nicole L.; McNally, Mark T.

    2008-01-01

    Polyadenylation of Rous sarcoma virus (RSV) RNA is inefficient, as approximately 15% of RSV RNAs represent read-through transcripts that use a downstream cellular polyadenylation site (poly(A) site). Read-through transcription has implications for the virus and the host since it is associated with oncogene capture and tumor induction. To explore the basis of inefficient RSV RNA 3'-end formation, we characterized RSV polyadenylation in vitro using HeLa cell nuclear extracts and HEK293 whole cell extracts. RSV polyadenylation substrates composed of the natural 3' end of viral RNA and various lengths of upstream sequence showed little or no polyadenylation, indicating that the RSV poly(A) site is suboptimal. Efficiently used poly(A) sites often have identifiable upstream and downstream elements (USEs and DSEs) in close proximity to the conserved AAUAAA signal. The sequences upstream and downstream of the RSV poly(A) site deviate from those found in efficiently used poly(A) sites, which may explain inefficient RSV polyadenylation. To assess the quality of the RSV USEs and DSEs, the well-characterized SV40 late USEs and/or DSEs were substituted for the RSV elements and vice versa, which showed that the USEs and DSEs from RSV are suboptimal but functional. CstF interacted poorly with the RSV polyadenylation substrate, and the inactivity of the RSV poly(A) site was at least in part due to poor CstF binding since tethering CstF to the RSV substrate activated polyadenylation. Our data are consistent with poor polyadenylation factor binding sites in both the USE and DSE as the basis for inefficient use of the RSV poly(A) site and point to the importance of additional elements within RSV RNA in promoting 3' end formation

  7. Single-site neural tube closure in human embryos revisited.

    Science.gov (United States)

    de Bakker, Bernadette S; Driessen, Stan; Boukens, Bastiaan J D; van den Hoff, Maurice J B; Oostra, Roelof-Jan

    2017-10-01

    Since the multi-site closure theory was first proposed in 1991 as explanation for the preferential localizations of neural tube defects, the closure of the neural tube has been debated. Although the multi-site closure theory is much cited in clinical literature, single-site closure is most apparent in literature concerning embryology. Inspired by Victor Hamburgers (1900-2001) statement that "our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right", we decided to critically review both theories of neural tube closure. To verify the theories of closure, we studied serial histological sections of 10 mouse embryos between 8.5 and 9.5 days of gestation and 18 human embryos of the Carnegie collection between Carnegie stage 9 (19-21 days) and 13 (28-32 days). Neural tube closure was histologically defined by the neuroepithelial remodeling of the two adjoining neural fold tips in the midline. We did not observe multiple fusion sites in neither mouse nor human embryos. A meta-analysis of case reports on neural tube defects showed that defects can occur at any level of the neural axis. Our data indicate that the human neural tube fuses at a single site and, therefore, we propose to reinstate the single-site closure theory for neural tube closure. We showed that neural tube defects are not restricted to a specific location, thereby refuting the reasoning underlying the multi-site closure theory. Clin. Anat. 30:988-999, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Site, Sector, Scope: Mapping the Epistemological Landscape of Health Humanities.

    Science.gov (United States)

    Charise, Andrea

    2017-12-01

    This essay presents a critical appraisal of the current state of baccalaureate Health Humanities, with a special focus on the contextual differences currently influencing the implementation of this field in Canada and, to a lesser extent, the United States and United Kingdom. I argue that the epistemological bedrock of Health Humanities goes beyond that generated by its written texts to include three external factors that are especially pertinent to undergraduate education: site (the setting of Health Humanities education), sector (the disciplinary eligibility for funding) and scope (the critical engagement with a program's local context alongside an emergent "core" of Health Humanities knowledge, learning, and practice). Drawing largely from the Canadian context, I discuss how these differences can inform or obstruct this field's development, and offer preliminary recommendations for encouraging the growth of baccalaureate Health Humanities-in Canada and elsewhere-in light of these factors.

  9. STRATEGI PEMECAHAN MASALAH MATEMATIS VERSI GEORGE POLYA DAN PENERAPANNYA DALAM PEMBELAJARAN MATEMATIKA

    Directory of Open Access Journals (Sweden)

    Wahid Umar

    2016-05-01

    Full Text Available George Polya telah meletakan suatu warisan “pentingnya mengajar dengan pemecahan masalah”. Setiap masalah memiliki “sepuluh strategi” yang tepat dengan “empat” langkah pemecahan sesuai dengan aspek-aspek dan sudut pandangnya masing-masing di dalam menyelesaikan suatu masalah matematis. Topik ini telah menjadi komponen utama dalam kurikulum matematika pada semua tingkatan pendidikan. NCTM dalam standards (1989 mempublikasikan ”The Curriculum and Evaluations Standards for School Mathematics”, yang menekankan bahwa pemecahan masalah harus menjadi fokus dalam kurikulum matematika di sekolah. Ini berarti bahwa pemecahan masalah merupakan salah satu topik yang sangat penting dalam pembelajaran matematika. Tujuan mengajarkan matematika dengan pemecahan masalah adalah: (1 membantu guru memperbaiki keterampilan pemecahan masalah diri sendiri; (2 diberikan kepada guru untuk membantu siswa mengembangkan keterampilan pemecahan masalah mereka; (3 untuk menyelidiki strategi umum pemecahan masalah; dan (4 bagaimana membuat kata “masalah” dan “pemecahan masalah” menantang dan menarik untuk siswa. Pentingnya para siswa mengalami proses pembelajaran matematika dengan pemecahan masalah matematis. Siswa perlu dipersiapkan dan didorong untuk berpikir bahwa sesuatu itu multi-dimensi sehingga mereka dapat melihat banyak kemungkinan penyelesaian untuk suatu masalah. Dengan demikian, pemecahan masalah matematis dalam pembelajaran matematika merupakan bagian integral dari semua aktivitas matematis. Fokus kajian makalah ini adalah bagaimana strategi pemecahan masalah matematis versi George Polya dan penerapannya dalam pembelajaran matematika.

  10. Host genetic variation impacts microbiome composition across human body sites.

    Science.gov (United States)

    Blekhman, Ran; Goodrich, Julia K; Huang, Katherine; Sun, Qi; Bukowski, Robert; Bell, Jordana T; Spector, Timothy D; Keinan, Alon; Ley, Ruth E; Gevers, Dirk; Clark, Andrew G

    2015-09-15

    The composition of bacteria in and on the human body varies widely across human individuals, and has been associated with multiple health conditions. While microbial communities are influenced by environmental factors, some degree of genetic influence of the host on the microbiome is also expected. This study is part of an expanding effort to comprehensively profile the interactions between human genetic variation and the composition of this microbial ecosystem on a genome- and microbiome-wide scale. Here, we jointly analyze the composition of the human microbiome and host genetic variation. By mining the shotgun metagenomic data from the Human Microbiome Project for host DNA reads, we gathered information on host genetic variation for 93 individuals for whom bacterial abundance data are also available. Using this dataset, we identify significant associations between host genetic variation and microbiome composition in 10 of the 15 body sites tested. These associations are driven by host genetic variation in immunity-related pathways, and are especially enriched in host genes that have been previously associated with microbiome-related complex diseases, such as inflammatory bowel disease and obesity-related disorders. Lastly, we show that host genomic regions associated with the microbiome have high levels of genetic differentiation among human populations, possibly indicating host genomic adaptation to environment-specific microbiomes. Our results highlight the role of host genetic variation in shaping the composition of the human microbiome, and provide a starting point toward understanding the complex interaction between human genetics and the microbiome in the context of human evolution and disease.

  11. Developing Hydrogeological Site Characterization Strategies based on Human Health Risk

    Science.gov (United States)

    de Barros, F.; Rubin, Y.; Maxwell, R. M.

    2013-12-01

    In order to provide better sustainable groundwater quality management and minimize the impact of contamination in humans, improved understanding and quantification of the interaction between hydrogeological models, geological site information and human health are needed. Considering the joint influence of these components in the overall human health risk assessment and the corresponding sources of uncertainty aid decision makers to better allocate resources in data acquisition campaigns. This is important to (1) achieve remediation goals in a cost-effective manner, (2) protect human health and (3) keep water supplies clean in order to keep with quality standards. Such task is challenging since a full characterization of the subsurface is unfeasible due to financial and technological constraints. In addition, human exposure and physiological response to contamination are subject to uncertainty and variability. Normally, sampling strategies are developed with the goal of reducing uncertainty, but less often they are developed in the context of their impacts on the overall system uncertainty. Therefore, quantifying the impact from each of these components (hydrogeological, behavioral and physiological) in final human health risk prediction can provide guidance for decision makers to best allocate resources towards minimal prediction uncertainty. In this presentation, a multi-component human health risk-based framework is presented which allows decision makers to set priorities through an information entropy-based visualization tool. Results highlight the role of characteristic length-scales characterizing flow and transport in determining data needs within an integrated hydrogeological-health framework. Conditions where uncertainty reduction in human health risk predictions may benefit from better understanding of the health component, as opposed to a more detailed hydrogeological characterization, are also discussed. Finally, results illustrate how different dose

  12. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  13. Presence of a polyA tail at the 3’-end of Maize rayado fino virus RNA

    Science.gov (United States)

    Maize rayado fino virus (MRFV) is the type member of the genus Marafivirus in the family Tymoviridae, yet is distinct from other members of the genus in that its genome reportedly lacks a poly(A) tail at the 3’-terminus. Using naïve and targeted PCR-based approaches, we now show that the MRFV genom...

  14. Human Mars Landing Site and Impacts on Mars Surface Operations

    Science.gov (United States)

    Hoffman, Stephen J.; Bussey, Ben

    2016-01-01

    This paper describes NASA's initial steps for identifying and evaluating candidate Exploration Zones (EZs) and Regions of Interests (ROIs) for the first human crews that will explore the surface of Mars. NASA's current effort to define the exploration of this planet by human crews, known as the Evolvable Mars Campaign (EMC), provides the context in which these EZs and ROIs are being considered. The EMC spans all aspects of a human Mars mission including launch from Earth, transit to and from Mars, and operations on the surface of Mars. An EZ is a collection of ROIs located within approximately 100 kilometers of a centralized landing site. ROIs are areas relevant for scientific investigation and/or development/maturation of capabilities and resources necessary for a sustainable human presence. The EZ also contains one or more landing sites and a habitation site that will be used by multiple human crews during missions to explore and utilize the ROIs within the EZ. With the EMC as a conceptual basis, the EZ model has been refined to a point where specific site selection criteria for scientific exploration and in situ resource utilization can be defined. In 2015 these criteria were distributed to the planetary sciences community and the in situ resource utilization and civil engineering communities as part of a call for EZ proposals. The resulting "First Landing Site/Exploration Zone Workshop for Human Missions to the Surface of Mars" was held in October 2015 during which 47 proposals for EZs and ROIs were presented and discussed. Proposed locations spanned all longitudes and all allowable latitudes (+/- 50 degrees). Proposed justification for selecting one of these EZs also spanned a significant portion of the scientific and resource criteria provided to the community. Several important findings resulted from this Workshop including: (a) a strong consensus that, at a scale of 100 km (radius), multiple places on Mars exist that have both sufficient scientific interest

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  16. Gender, health, and human rights in sites of political exclusion.

    Science.gov (United States)

    Laurie, M; Petchesky, R P

    2008-01-01

    In this paper, we investigate the intersections of gender, health and human rights in sites of political exclusion. We apply the political theory of Giorgio Agamben on 'states of exception', seeking to better understand how the recent 'war on terror', that seemingly knows no limits of time or space, is driving health outcomes in refugee and Internally Displaced Persons (IDP) camps. Reproductive health, militarization, and gender-based violence in camps are explored in depth. The evidence presented reveals a number of contradictions of refugee and IDP camps, further highlighting the need for a more rights based humanitarianism. We conclude that foregrounding states of exception, as a way of understanding current gender dynamics in the social determinants of health, is both epidemiologically necessary and conceptually useful. We find that, in these sites of exclusion, the indispensability of a human rights approach to gender and health equity issues is revealed most directly. Furthermore, we are able to make new connections between the 'crisis of humanitarianism', gender, and health.

  17. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Merkert, Sylvia; Martin, Ulrich

    2016-06-24

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  18. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  19. A Simple Decision Rule for Recognition of Poly(A) Tail Signal Motifs in Human Genome

    KAUST Repository

    AbouEisha, Hassan M.; Chikalov, Igor; Moshkov, Mikhail; Jankovic, Boris R.

    2015-01-01

    Background is the numerous attempts were made to predict motifs in genomic sequences that correspond to poly (A) tail signals. Vast portion of this effort has been directed to a plethora of nonlinear classification methods. Even when such approaches

  20. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  1. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-01-01

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  2. Differential regulation of the human progesterone receptor gene through an estrogen response element half site and Sp1 sites.

    Science.gov (United States)

    Petz, Larry N; Ziegler, Yvonne S; Schultz, Jennifer R; Kim, Hwajin; Kemper, J Kim; Nardulli, Ann M

    2004-02-01

    The progesterone receptor (PR) gene is regulated by estrogen in normal reproductive tissues and in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated by interaction of the ligand-occupied estrogen receptor (ER) with estrogen response elements (EREs) in target genes, the human progesterone receptor (PR) gene lacks a palindromic ERE. Promoter A of the PR gene does, however, contain an ERE half site upstream of two adjacent Sp1 sites from +571 to +595, the +571 ERE/Sp1 site. We have examined the individual contributions of the ERE half site and the two Sp1 sites in regulating estrogen responsiveness. Transient transfection assays demonstrated that both Sp1 sites were critical for estrogen-mediated activation of the PR gene. Interestingly, rather than decreasing transcription, mutations in the ERE half site increased transcription substantially suggesting that this site plays a role in limiting transcription. Chromatin immunoprecipitation assays demonstrated that Sp1 was associated with the +571 ERE/Sp1 site in the endogenous PR gene in the absence and in the presence of estrogen, but that ERalpha was only associated with this region of the PR gene after MCF-7 cells had been treated with estrogen. Our studies provide evidence that effective regulation of transcription through the +571 ERE/Sp1 site requires the binding of ERalpha and Sp1 to their respective cis elements and the appropriate interaction of ERalpha and Sp1 with other coregulatory proteins and transcription factors.

  3. Nonenzymatic glycosylation of human hemoglobin at multiple sites

    International Nuclear Information System (INIS)

    Shapiro, R.; McManus, M.; Garrick, L.; McDonald, M.J.; Bunn, H.F.

    1979-01-01

    The most abundant minor hemoglobin component of human hemolysate is Hb A1c, which has glucose bound to the N-terminus of the beta chain by a ketoamine linkage. Hb A1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A1a1 and Hb A1a2 are adducts of sugar phosphates at the N-terminus of the beta chain. Hb A1b contains an unidentified nonphosphorylated sugar at the beta N-terminus. In addition, a significant portion of the major hemoglobin component (Hb Ao) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the alpha chain and the epsilon-amino group of several lysine residues on both the alpha and the beta chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion

  4. Human population and activities in Forsmark. Site description

    Energy Technology Data Exchange (ETDEWEB)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km{sup 2} near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the

  5. Human population and activities in Forsmark. Site description

    International Nuclear Information System (INIS)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km 2 near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the parish has been

  6. Human resources FY 1995 Site Program Plan WBS 6.10.2

    Energy Technology Data Exchange (ETDEWEB)

    1994-09-01

    This document contains information concerning human resources management at the Hanford Reservation. Information discusses the following topics: Cost estimates, closure and placement of labor resources, and management of human resources throughout the Hanford Site.

  7. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A tail at the 3'-end.

    Directory of Open Access Journals (Sweden)

    Shushan Harutyunyan

    Full Text Available Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.

  8. Gains of ubiquitylation sites in highly conserved proteins in the human lineage

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.

  9. Human Splice-Site Prediction with Deep Neural Networks.

    Science.gov (United States)

    Naito, Tatsuhiko

    2018-04-18

    Accurate splice-site prediction is essential to delineate gene structures from sequence data. Several computational techniques have been applied to create a system to predict canonical splice sites. For classification tasks, deep neural networks (DNNs) have achieved record-breaking results and often outperformed other supervised learning techniques. In this study, a new method of splice-site prediction using DNNs was proposed. The proposed system receives an input sequence data and returns an answer as to whether it is splice site. The length of input is 140 nucleotides, with the consensus sequence (i.e., "GT" and "AG" for the donor and acceptor sites, respectively) in the middle. Each input sequence model is applied to the pretrained DNN model that determines the probability that an input is a splice site. The model consists of convolutional layers and bidirectional long short-term memory network layers. The pretraining and validation were conducted using the data set tested in previously reported methods. The performance evaluation results showed that the proposed method can outperform the previous methods. In addition, the pattern learned by the DNNs was visualized as position frequency matrices (PFMs). Some of PFMs were very similar to the consensus sequence. The trained DNN model and the brief source code for the prediction system are uploaded. Further improvement will be achieved following the further development of DNNs.

  10. Human health and other risk drivers to prioritize site remediation

    Energy Technology Data Exchange (ETDEWEB)

    McHugh, T.; Connor, J. [Groundwater Services Inc, Houston, TX (United States)

    2003-07-01

    Remedial actions at soil and groundwater cleanup sites have traditionally been addressed on an individual, case-by-case basis, as needed to address regulatory requirements. However, effective management of large portfolios of remediation sites (such as hundreds or thousands of underground storage tank sites owned by a single company) requires coordination and prioritisation of individual site response actions to optimise the degree of risk reduction achieved with available resources. To meet these management objectives, two new risk-based management tools have been developed and implemented by the authors: i) a simple risk-based classification system, that can be employed to prioritise response actions, identify key risk drivers, and measure risk reduction progress over time for the full site portfolio; and ii) a lifecycle cost management system that can be employed to forecast remediation spending and optimise risk reduction benefits. For use in prioritising response actions at remediation sites, 'risk' is defined as the negative consequence of no action. (orig.)

  11. Human population and activities at Simpevarp. Site description

    International Nuclear Information System (INIS)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km 2 near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km 2 , three times lower than in Kalmar laen. The demography statistics show

  12. Human population and activities at Simpevarp. Site description

    Energy Technology Data Exchange (ETDEWEB)

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  13. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites and...

  14. Towards a protocol for the assessment of site-specific human health risks for consumption of vegetables from contaminated sites

    NARCIS (Netherlands)

    Swartjes FA; Dirven-van Breemen EM; Otte PF; Beelen P van; Rikken MGJ; Tuinstra J; Spijker J; Lijzen JPA; LER

    2007-01-01

    RIVM has developed an approach which allows human health risks of vegetable consumption from contaminated sites to be assessed. A tiered approach was used to guarantee the scientific basis and efficient use in practice. The underlying principle is: simple when possible and complex when necessary. If

  15. The distribution of lectin receptor sites in human breast lesions.

    Science.gov (United States)

    Skutelsky, E; Hoenig, S; Griffel, B; Alroy, J

    1988-08-01

    Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.

  16. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    Science.gov (United States)

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  17. Systematic profiling of poly(A+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

    Directory of Open Access Journals (Sweden)

    Wencheng Li

    2015-04-01

    Full Text Available Alternative cleavage and polyadenylation (APA results in mRNA isoforms containing different 3' untranslated regions (3'UTRs and/or coding sequences. How core cleavage/polyadenylation (C/P factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A sites (pAs, CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

  18. PROSES BERPIKIR SISWA SEKOLAH MENENGAH PERTAMA DALAM MEMECAHKAN MASALAH MATEMATIKA BERDASARKAN LANGKAH-LANGKAH POLYA DITINJAU DARI ADVERSITY QUOTIENT

    Directory of Open Access Journals (Sweden)

    Muhammad Yani

    2016-06-01

    Full Text Available Penelitian ini dilakukan untuk menjelaskan proses berpikir dan menganalisis kesulitan siswa dalam memecahkan masalah matematika berdasarkan pengukuran Polya ditinjau dari Adversity Quotient (AQ. Penelitian ini merupakan penelitian deskirptif kualitatif dengan subjek penelitian adalah siswa dari kelas IX SMP N 1 Banda Aceh tediri dari tiga siswa. Pemilihan subjek penelitian menggunakan metode purposive sampling dan berdasarkan tingkatan AQ (climber, camper, dan quitter dan komunikasi (lisan dan tertulis. Pengumpulan data menggunakan wawancara berbasis tugas, dan triangulasi untuk mengecek validitas data. Data dianalisis menggunakan konsep dari Miles dan Huberman: yaitu tahap pengurangan data, presentasi data, dan kesimpulan. Hasil menunjukkan bahwa: (1 Proses berpikir dari subjek climber yaitu secara asimilasi dalam memahami, merencanakan penyelesaian, .serta mengecek kembali; (2 Subjek camper juga berpikir secara asimilasi pada tahap memahami masalah, merencanakan penyelesaian, dan mengecek kembali; (3 subjek quitter berpikir secara akomodasi dalam memahami masalah dan menyelesaikan masalah. Kata kunci: Proses Berpikir, Pemecahan Masalah, Tahap Polya, Adversity Quotient (AQ DOI: http://dx.doi.org/10.22342/jpm.10.1.3278.42-57

  19. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    International Nuclear Information System (INIS)

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-01-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125 I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells

  20. Identification of Polyadenylation Sites within Arabidopsis Thaliana

    KAUST Repository

    Kalkatawi, Manal

    2011-09-01

    Machine Learning (ML) is a field of artificial intelligence focused on the design and implementation of algorithms that enable creation of models for clustering, classification, prediction, ranking and similar inference tasks based on information contained in data. Many ML algorithms have been successfully utilized in a variety of applications. The problem addressed in this thesis is from the field of bioinformatics and deals with the recognition of polyadenylation (poly(A)) sites in the genomic sequence of the plant Arabidopsis thaliana. During the RNA processing, a tail consisting of a number of consecutive adenine (A) nucleotides is added to the terminal nucleotide of the 3’- untranslated region (3’UTR) of the primary RNA. The process in which these A nucleotides are added is called polyadenylation. The location in the genomic DNA sequence that corresponds to the start of terminal A nucleotides (i.e. to the end of 3’UTR) is known as a poly(A) site. Recognition of the poly(A) sites in DNA sequence is important for better gene annotation and understanding of gene regulation. In this study, we built an artificial neural network (ANN) for the recognition of poly(A) sites in the Arabidopsis thaliana genome. Our study demonstrates that this model achieves improved accuracy compared to the existing predictive models for this purpose. The key factor contributing to the enhanced predictive performance of our ANN model is a distinguishing set of features used in creation of the model. These features include a number of physico-chemical characteristics of relevance, such as dinucleotide thermodynamic characteristics, electron-ion interaction potential, etc., but also many of the statistical properties of the DNA sequences from the region surrounding poly(A) site, such as nucleotide and polynucleotide properties, common motifs, etc. Our ANN model was compared in performance with several other ML models, as well as with the PAC tool that is specifically developed for

  1. A tool for improving the management of social and human rights risks at project sites: The Human Rights Sphere

    NARCIS (Netherlands)

    van der Ploeg, Lidewij; Vanclay, Frank

    2017-01-01

    This paper identifies and addresses the challenges of implementing the corporate responsibility to respect human rights in practice at project sites. To support on-ground operational staff, we offer the Human Rights Sphere (HRS), a practical tool we developed from empirical research in three

  2. A tool for improving the management of social and human rights risks at project sites : The Human Rights Sphere

    NARCIS (Netherlands)

    van der Ploeg, Lidewij; Vanclay, Frank

    2017-01-01

    This paper identifies and addresses the challenges of implementing the corporate responsibility to respect human rights in practice at project sites. To support on-ground operational staff, we offer the Human Rights Sphere (HRS), a practical tool we developed from empirical research in three

  3. Characterization of reference and site specific human acids

    International Nuclear Information System (INIS)

    Kim, J.I.; Buckau, G.

    1988-01-01

    As a part of the interlaboratory exercise for the complexation of humic acid and colloid generation (COCO-Club activities) in the CEC project MIRAGE-II, the characterization of humic acids have been carried out, as for their elemental compositions, inorganic impurities, spectroscopic properties, size distributions and proton exchange capacities. The commercial humic acid (Na salt) from Aldrich Co. is purified to a protonated form and used as a reference material, and the humic acid extracted from one of Gorleben groundwaters is also purified to a protonated form and taken as a site specific material. These two humic acids, together with the original Na salt from Aldrich Co., are included for the characterization exercise. The results of characterization provide a basic knowledge that supports the forthcoming study of complexation of humic acids with actinides and fission products in their migration processes in the geosphere. (orig.)

  4. Saccharomyces cerevisiae Ngl3p is an active 3′–5′ exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

    DEFF Research Database (Denmark)

    Feddersen, Ane; Dedic, Emil; Poulsen, Esben Guldahl

    2012-01-01

    RNAs that yeast Ngl3p is a functional 3′–5′ exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p...

  5. Intramolecular cyclization in irradiated nucleic acids: correlation between high-performance liquid chromatography and an immunochemical assay for 8,5'-cycloadenosine in irradiated poly(A)

    International Nuclear Information System (INIS)

    Fuciarelli, A.F.; Shum, F.Y.; Raleigh, J.A.

    1987-01-01

    A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions

  6. Factors for assessment of human health risk associated with remedial action at hazardous waste sites

    International Nuclear Information System (INIS)

    Stephenson, D.E.; King, C.M.; Looney, B.B.; Holmes, W.G.; Gordon, D.E.

    1985-01-01

    A risk assessment strategy that is cost effective and minimized human health risks was developed for closure of hazardous waste sites at the Savannah River Plant. The strategy consists of (1) site characterization, (2) contaminant transport modeling, and (3) determination of relative merits of alternative remedial actions according to the degree of health protection they provide

  7. Molecular cloning and characterization of a novel isoform of the non-canonical poly(A) polymerase PAPD7

    Energy Technology Data Exchange (ETDEWEB)

    Ogami, Koichi; Cho, Rihe [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Hoshino, Shin-ichi, E-mail: hoshino@phar.nagoya-cu.ac.jp [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)

    2013-03-01

    Highlights: ► So far, only an enzymatically inactive isoform of PAPD7 was reported. ► The novel isoform: PAPD7 l shows robust nucleotidyl transferase activity. ► The newly identified amino terminal region is required for the activity. ► PAPD7 l localizes to the nucleoplasm. ► The N terminal region identified is also required for the nuclear localization. - Abstract: Non-canonical poly(A) polymerases (ncPAPs) catalyze the addition of poly(A) tail to the 3′ end of RNA to play pivotal roles in the regulation of gene expression and also in quality control. Here we identified a novel isoform of the 7th member of ncPAPs: PAPD7 (PAPD7 l), which contains 230 extra amino acids at the amino terminus of the previously identified PAPD7 (PAPD7 s). In sharp contrast to the inactive PAPD7 s, PAPD7 l showed robust nucleotidyl transferase activity when tethered to an RNA. A region required for the activity was localized to 187–219 aa, and this region was also required for the nuclear retention of PAPD7 l. Western blot analysis revealed that 94 kDa band (corresponding to PAPD7 l) but not 62 kDa band (corresponding to PAPD7 s) detected by PAPD7 antibody was specifically depleted by treatment with PAPD7 siRNA in both HeLa and U2OS cells. These results suggest that PAPD7 l is the major and active isoform of PAPD7 expressed in cells.

  8. Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14

    International Nuclear Information System (INIS)

    Duechler, M.; Skern, T.; Sommergruber, W.; Neubauer, C.; Gruendler, P.; Fogy, I.; Blaas, D.; Kuechler, E.

    1987-01-01

    The complete nucleotide sequence of the genome of human rhinovirus type 89 was determined from the cDNA that had been cloned into Escherichia coli. The genome is 7152 nucleotides long and contains a single large open reading frame of 2164 codons. Translation commences at position 619 and ends 42 nucleotides before the poly(a) tract. The positions of three proteolytic cleavage sites in the polyprotein were determined by N-terminal amino acid sequencing of the capsid proteins; the remainder were predicted from comparisons with other picornaviruses. Extensive similarity between the derived amino acid sequences of human rhinovirus types 89 and 2 was found, whereas the similarity between human rhinovirus types 89 and 14 was considerably less. It is apparent that human rhinoviruses may be more closely related than has been previously thought

  9. A comparative study of microbial diversity and community structure in marine sediments using poly(A tailing and reverse transcription PCR

    Directory of Open Access Journals (Sweden)

    Tatsuhiko eHoshino

    2013-06-01

    Full Text Available To obtain a better understanding of metabolically active microbial communities, we tested a molecular ecological approach using poly(A tailing of environmental 16S rRNA, followed by full-length complementary DNA (cDNA synthesis and sequencing to eliminate potential biases caused by mismatching of PCR primer sequences. The RNA pool tested was extracted from marine sediments of the Yonaguni Knoll IV hydrothermal field in the southern Okinawa Trough. The sequences obtained using the ploy(A tailing method were compared statistically and phylogenetically with those obtained using conventional reverse transcription-polymerase chain reaction (RT-PCR with published domain-specific primers. Both methods indicated that Deltaproteobacteria are predominant in sediment (>85% of the total sequence read. The poly(A tailing method indicated that Desulfobacterales were the predominant deltaproteobacteria, while most of the sequences in libraries constructed using RT-PCR were derived from Desulfuromonadales. This discrepancy may have been due to low coverage of Desulfobacterales by the primers used. A comparison of library diversity indices indicated that the poly(A tailing method retrieves more phylogenetically diverse sequences from the environment. The four archaeal 16S rRNA sequences that were obtained using the poly(A tailing method formed deeply branching lineages that were related to Candidatus Parvarchaeum and the Ancient Archaeal Group. These results clearly demonstrate that poly(A tailing followed by cDNA sequencing is a powerful and less biased molecular ecological approach for the study of metabolically active microbial communities.

  10. Influence of nucleotide modifications at the C2' position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA.

    Science.gov (United States)

    Copp, William; Denisov, Alexey Y; Xie, Jingwei; Noronha, Anne M; Liczner, Christopher; Safaee, Nozhat; Wilds, Christopher J; Gehring, Kalle

    2017-09-29

    Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Site-specific semisynthetic variant of human hemoglobin

    International Nuclear Information System (INIS)

    Hefta, S.A.; Lyle, S.B.; Busch, M.R.; Harris, D.E.; Matthew, J.B.; Gurd, F.R.N.

    1988-01-01

    A single round of Edman degradation was employed to remove the NH 2 -terminal valine from isolated α chains of human hemoglobin. Reconstitution of normal β chains with truncated or substituted α chains was used to form truncated (des-Val 1 -α1) and substituted ([[1- 13 C]Gly 1 ]α1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val 1 -α chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-biphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the α-chain NH 2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH 2 -terminal pK/sub 1/2/ value was determined for the [ 13 C]glycine-substituted analog to be 7.46 +/- 0.09 at 15 0 C in the carbon monoxide-liganded form. This value, measured directly by 13 C NMR, agrees with the determination made by the less-direct 13 CO 2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is consistent with the presence of an NH 2 -terminal salt bridge to the carboxylate of Arg-141 of the α chain in the liganded form

  12. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes.

    Science.gov (United States)

    Qian, Wei; Wang, Yong; Li, Rui-Fu; Zhou, Xin; Liu, Jing; Peng, Dai-Zhi

    2017-03-03

    BACKGROUND Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. MATERIAL AND METHODS In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. RESULTS The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. CONCLUSIONS This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.

  13. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

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

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

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

  15. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  16. Nucleotide sequences of cDNAs for human papillomavirus type 18 transcripts in HeLa cells

    International Nuclear Information System (INIS)

    Inagaki, Yutaka; Tsunokawa, Youko; Takebe, Naoko; Terada, Masaaki; Sugimura, Takashi; Nawa, Hiroyuki; Nakanishi, Shigetada

    1988-01-01

    HeLa cells expressed 3.4- and 1.6-kilobase (kb) transcripts of the integrated human papillomavirus (HPV) type 18 genome. Two types of cDNA clones representing each size of HPV type 18 transcript were isolated. Sequence analysis of these two types of cDNA clones revealed that the 3.4-kb transcript contained E6, E7, the 5' portion of E1, and human sequence and that the 1.6-kb transcript contained spliced and frameshifted E6 (E6 * ), E7, and human sequence. There was a common human sequence containing a poly(A) addition signal in the 3' end portions of both transcripts, indicating that they were transcribed from the HPV genome at the same integration site with different splicing. Furthermore, the 1.6-kb transcript contained both of the two viral TATA boxes upstream of E6, strongly indicating that a cellular promoter was used for its transcription

  17. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

    Science.gov (United States)

    Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

    2018-02-01

    Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

  18. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available Recently, the clustered regularly interspaced short palindromic repeats (CRISPR system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

  19. The first trimester human placenta is a site for terminal maturation of primitive erythroid cells

    OpenAIRE

    Van Handel, Ben; Prashad, Sacha L.; Hassanzadeh-Kiabi, Nargess; Huang, Andy; Magnusson, Mattias; Atanassova, Boriana; Chen, Angela; Hamalainen, Eija I.; Mikkola, Hanna K. A.

    2010-01-01

    Embryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryo's immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in fir...

  20. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  1. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    Science.gov (United States)

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  2. Plutonium-aerosol emission rates and human pulmonary deposition calculations for Nuclear Site 201, Nevada Test Site

    International Nuclear Information System (INIS)

    Shinn, J.H.; Homan, D.N.

    1982-01-01

    This study determined the plutonium-aerosol fluxes from the soil to quantify (1) the extent of potential human exposure by deep-lung retention of alpha-emitting particles; (2) the source term should there be any significant, long-term, transport of plutonium aerosols; and (3) the resuspension factor and rate so that, for the first time at any nuclear site, one may calculate how long it will take for wind erosion to carry away a significant amount of the contaminated soil. High-volume air samplers and cascade impactors were used to characterize the plutonium aerosols. Meteorological flux-profile methods were used to calculate dust and plutonium aerosol emission rates. A floorless wind tunnel (10-m long) was used to examine resuspension under steady-state, high wind speed. The resuspension factor was two orders of magnitude lower than the other comparable sites at NTS and elsewhere, and the average resuspension rate of 5.3 x 10 -8 /d was also very low, so that the half-time for resuspension by wind erosion was about 36,000 y

  3. Human Health and Ecological Risk Assessment Work Plan Mud Pit Release Sites, Amchitka Island, Alaska

    Energy Technology Data Exchange (ETDEWEB)

    DOE/NV

    2001-03-12

    This Work Plan describes the approach that will be used to conduct human health and ecological risk assessments for Amchitka Island, Alaska, which was utilized as an underground nuclear test site between 1965 and 1971. During this period, the U.S. Atomic Energy Commission (now the U.S. Department of Energy) conducted two nuclear tests (known as Long Shot and Milrow) and assisted the U.S. Department of Defense with a third test (known as Cannikin). Amchitka Island is approximately 42 miles long and located 1,340 miles west-southwest of Anchorage, Alaska, in the western end of the Aleutian Island archipelago in a group of islands known as the Rat Islands. Historically including deep drilling operations required large volumes of drilling mud, a considerable amount of which was left on the island in exposed mud pits after testing was completed. Therefore, there is a need for drilling mud pit remediation and risk assessment of historical mud pit releases. The scope of this work plan is to document the environmental objectives and the proposed technical site investigation strategies that will be utilized for the site characterization of the constituents in soil, surface water, and sediment at these former testing sites. Its goal is the collection of data in sufficient quantity and quality to determine current site conditions, support a risk assessment for the site surfaces, and evaluate what further remedial action is required to achieve permanent closure of these three sites that will protect both human health and the environment. Suspected compounds of potential ecological concern for investigative analysis at these sites include diesel-range organics, polyaromatic hydrocarbons, polychlorinated biphenyls, volatile organic compounds, and chromium. The results of these characterizations and risk assessments will be used to evaluate corrective action alternatives to include no further action, the implementation of institutional controls, capping on site, or off-sit e

  4. Confluence of arts, humanities, and science at sites of long-term ecological inquiry

    Science.gov (United States)

    Frederick J. Swanson

    2015-01-01

    Over the past century, ecology, the arts, and humanities diverged, but are now converging again, especially at sites of long-term, place-based ecological inquiry. This convergence has been inspired in part by the works of creative, boundary-spanning individuals and the long-standing examples of artshumanities programs in intriguing landscapes, such as artist and writer...

  5. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

    Energy Technology Data Exchange (ETDEWEB)

    Tomkinson, B.; Wernstedt, C.; Hellman, U.; Zetterqvist, Oe.

    1987-11-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.

  6. A framework of dynamic analysis for risks asociated with human activity at radioactive waste disposal sites

    International Nuclear Information System (INIS)

    Umeki, H.; Masuda, S.

    1989-01-01

    Human intrusive actions at radioactive waste disposal sites which cause radiological impact were identified in connection with other events and processes on the influence diagram. The scenarios of less likely events including human intrusive actions were generated from the diagram and then treated by probabilistic way. For assigning probabilities of events dynamically, simultaneous difference equations were introduced and simple general solutions which satisfy the equations were used to illustrate the example calculations for both direct and indirect impacts by human intrusive actions such as drilling/mining and pumping of groundwater. The method developed here will be useful for both scenario screening in overall scenario study and risk calculation combined with consequence analysis

  7. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

  8. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  9. 3ircular dichroism simulation shows a site-II-to-site-I displacement of human serum albumin-bound diclofenac by ibuprofen

    OpenAIRE

    Yamasaki, Keishi; Rahman, Mohammed Hablbur; Tsutsumi, Yasuhiro; Maruyama, Toru; Ahmed, Shamim; Kragh-Hansen; Otagiri, Masaki

    2000-01-01

    Purpose: The purpose of this study was to confirm the hypothesis that a site-II-to-site-I displacement takes place when some nonsteroidal anti-inflammatory drugs are displaced by another drug from their high-affinity binding site to a site of lower affinity on human serum albumin (HSA).Methods: Diclofenac, sodium salt, was used as a representative example because of its prominent reversal of the Cotton effect. Effects of site-specific drugs on the free fraction of diclofenac were determined b...

  10. Savannah River Site human error data base development for nonreactor nuclear facilities

    International Nuclear Information System (INIS)

    Benhardt, H.C.; Held, J.E.; Olsen, L.M.; Vail, R.E.; Eide, S.A.

    1994-01-01

    As part of an overall effort to upgrade and streamline methodologies for safety analyses of nonreactor nuclear facilities at the Savannah River Site (SRS), a human error data base has been developed and is presented in this report. The data base fulfills several needs of risk analysts supporting safety analysis report (SAR) development. First, it provides a single source for probabilities or rates for a wide variety of human errors associated with the SRS nonreactor nuclear facilities. Second, it provides a documented basis for human error probabilities or rates. And finally, it provides actual SRS-specific human error data to support many of the error probabilities or rates. Use of a single, documented reference source for human errors, supported by SRS-specific human error data, will improve the consistency and accuracy of human error modeling by SRS risk analysts. It is envisioned that SRS risk analysts will use this report as both a guide to identifying the types of human errors that may need to be included in risk models such as fault and event trees, and as a source for human error probabilities or rates. For each human error in this report, ffime different mean probabilities or rates are presented to cover a wide range of conditions and influencing factors. The ask analysts must decide which mean value is most appropriate for each particular application. If other types of human errors are needed for the risk models, the analyst must use other sources. Finally, if human enors are dominant in the quantified risk models (based on the values obtained fmm this report), then it may be appropriate to perform detailed human reliability analyses (HRAS) for the dominant events. This document does not provide guidance for such refined HRAS; in such cases experienced human reliability analysts should be involved

  11. mRNA decay proteins are targeted to poly(A+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Itzel López-Rosas

    Full Text Available In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies. In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i EhCAF1 colocalized with poly(A(+ RNA and (ii during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P

  12. Comparing the Scoring of Human Decomposition from Digital Images to Scoring Using On-site Observations.

    Science.gov (United States)

    Dabbs, Gretchen R; Bytheway, Joan A; Connor, Melissa

    2017-09-01

    When in forensic casework or empirical research in-person assessment of human decomposition is not possible, the sensible substitution is color photographic images. To date, no research has confirmed the utility of color photographic images as a proxy for in situ observation of the level of decomposition. Sixteen observers scored photographs of 13 human cadavers in varying decomposition stages (PMI 2-186 days) using the Total Body Score system (total n = 929 observations). The on-site TBS was compared with recorded observations from digital color images using a paired samples t-test. The average difference between on-site and photographic observations was -0.20 (t = -1.679, df = 928, p = 0.094). Individually, only two observers, both students with human decomposition based on digital images can be substituted for assessments based on observation of the corpse in situ, when necessary. © 2017 American Academy of Forensic Sciences.

  13. Elephants and human intervention in the Lower and Middle Pleistocene sites of Africa and Europe

    Directory of Open Access Journals (Sweden)

    Martos Romero, Juan Antonio

    1998-06-01

    Full Text Available In this paper, we review some African and European sites at which it is claimed that elephants were exploted by humans. The differences between sites with only one individual (type 1 and sites with a large number of elephants (type 2 are related to different formation processes and particular problems which limit the informative potential of sites.

    En el presente artículo se revisan yacimientos africanos y europeos donde se ha planteado la existencia de una intervención humana sobre elefantes. Se establece una diferencia entre sitios con un sólo individuo (tipo 1 o un número alto de elefantes (tipo 2. Con independencia del carácter de la intervención humana, cuando ésta es evidente, tales diferencias responden a unos procesos de formación distintos con problemáticas singulares que condicionan finalmente la capacidad informativa del yacimiento.

  14. The distribution of iron between the metal-binding sites of transferrin human serum.

    Science.gov (United States)

    Williams, J; Moreton, K

    1980-02-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.

  15. Amplification of a transcriptionally active DNA sequence in the human brain

    International Nuclear Information System (INIS)

    Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

    1986-01-01

    The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

  16. Risks to humans and wildlife from metal contamination in soils/sediments at CERCLA sites

    International Nuclear Information System (INIS)

    Hitch, J.P.; Hovatter, P.S.; Opresko, D.M.; Sample, B.; Young, R.A.

    1994-01-01

    A common problem that occurs at DOD and DOE CERCLA sites is metal contamination in soils and aquatic sediments and the protection of humans and wildlife from potential exposure to this contamination. Consequently, the authors have developed a site-specific reference dose for mercury in sediments at the Oak Ridge Reservation and site-specific cleanup levels for certain metals, including arsenic and nickel, in soils at an Army ammunition plant. Another concern during remediation of these sites is that limited data are available to determine the direct risks to indigenous wildlife. Therefore, the authors have developed toxicological benchmarks for certain metals and metal compounds to be used as screening tools to determine the potential hazard of a contaminant to representative mammalian and avian wildlife species. These values should enable the Army and DOE to more accurately determine the risks to humans and wildlife associated with exposure to these contaminated media at their sites in order to achieve a more effective remediation. This effort is ongoing at ORNL with toxicological benchmarks also being developed for metal compounds and other chemicals of concern to DOD and DOE in order to address the potential hazard to

  17. Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

    Energy Technology Data Exchange (ETDEWEB)

    Shima, K.; Kitayama, S.; Nakano, R.

    1987-05-01

    Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3 beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.

  18. Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

    Energy Technology Data Exchange (ETDEWEB)

    Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

    2017-11-22

    Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

  19. Omni-PolyA: a method and tool for accurate recognition of Poly(A) signals in human genomic DNA

    KAUST Repository

    Magana-Mora, Arturo; Kalkatawi, Manal M.; Bajic, Vladimir B.

    2017-01-01

    several machine learning techniques such as different classifiers in a tree-like decision structure and genetic algorithms for deriving a robust classification model. We performed a comparison between results obtained by state-of-the-art methods, deep

  20. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

    Science.gov (United States)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

    2012-09-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

  1. Low nucleosome occupancy is encoded around functional human transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Daenen Floris

    2008-07-01

    Full Text Available Abstract Background Transcriptional regulation of genes in eukaryotes is achieved by the interactions of multiple transcription factors with arrays of transcription factor binding sites (TFBSs on DNA and with each other. Identification of these TFBSs is an essential step in our understanding of gene regulatory networks, but computational prediction of TFBSs with either consensus or commonly used stochastic models such as Position-Specific Scoring Matrices (PSSMs results in an unacceptably high number of hits consisting of a few true functional binding sites and numerous false non-functional binding sites. This is due to the inability of the models to incorporate higher order properties of sequences including sequences surrounding TFBSs and influencing the positioning of nucleosomes and/or the interactions that might occur between transcription factors. Results Significant improvement can be expected through the development of a new framework for the modeling and prediction of TFBSs that considers explicitly these higher order sequence properties. It would be particularly interesting to include in the new modeling framework the information present in the nucleosome positioning sequences (NPSs surrounding TFBSs, as it can be hypothesized that genomes use this information to encode the formation of stable nucleosomes over non-functional sites, while functional sites have a more open chromatin configuration. In this report we evaluate the usefulness of the latter feature by comparing the nucleosome occupancy probabilities around experimentally verified human TFBSs with the nucleosome occupancy probabilities around false positive TFBSs and in random sequences. Conclusion We present evidence that nucleosome occupancy is remarkably lower around true functional human TFBSs as compared to non-functional human TFBSs, which supports the use of this feature to improve current TFBS prediction approaches in higher eukaryotes.

  2. Pyrimidine dimer sites associated with the daughter DNA strands in uv-irradiated human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, A R; Kirk-Bell, S [Sussex Univ., Brighton (UK)

    1978-03-01

    Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in uv-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing uv-specific endonuclease activity. In DNA synthesized immediately after irradiation, the frequency of these daughter strand dimer sites was 7 to 20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between uv irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following uv irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed.

  3. Pyrimidine dimer sites associated with the daughter DNA strands in UV-irradiated human fibroblasts

    International Nuclear Information System (INIS)

    Lehmann, A.R.; Kirk-Bell, S.

    1978-01-01

    Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in UV-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing UV-specific endonuclease activity. In DNA synthesized immediately after irradiation the frequency of these daughter strand dimer sites was 7-20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between UV-irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following UV-irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed. (author)

  4. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    Energy Technology Data Exchange (ETDEWEB)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-09-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples.

  5. Autoradiographic quantification of vasoactive intestinal peptide binding sites in sections from human blood mononuclear cell pellets

    International Nuclear Information System (INIS)

    Gutkind, J.S.; Kurihara, M.; Castren, E.; Saavedra, J.M.

    1988-01-01

    Quantitative autoradiographic methods were utilized to characterize specific, high-affinity vasoactive intestinal peptide binding sites (Kd = 310 +/- 60 pmol/L; Bmax = 93 +/- 11 fmol/mg protein) in frozen sections obtained from a mononuclear cell pellet derived from 20 ml of human blood. The method is at least one order of magnitude more sensitive than conventional membrane binding techniques, and it has the potential for wide applications in studies of neuropeptide, biogenic amine, and drug binding in clinical samples

  6. The Influence of Social Networking Sites on Recruiting Human Resources in the Czech Republic

    Directory of Open Access Journals (Sweden)

    Bohmova Lucie

    2015-02-01

    Full Text Available Background: This paper is focused on the usage of social networking sites (SNS for human resources departments in the process of hiring new employees. It also maps the development and influence of SNS on recruiter's behavior and customs. The main aim is to find out, whether SNS could/will replace traditional online job boards in the Czech Republic. The motivation for the research is to determine whether SNS can be used for serious and practical business purposes.

  7. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  8. Potential for effects of land contamination on human health. 2. The case of waste disposal sites.

    Science.gov (United States)

    Kah, Melanie; Levy, Len; Brown, Colin

    2012-01-01

    This review of the epidemiological literature shows that evidence for negative impacts of land contaminated by waste disposal on human health is limited. However, the potential for health impacts cannot be dismissed. The link between residence close to hazardous waste disposal sites and heightened levels of stress and anxiety is relatively well established. However, studies on self-reported outcomes generally suffer from interpretational problems, as subjective symptoms may be due to increased perception and recall. Several recent multiple-site studies support a plausible linkage between residence near waste disposal sites and reproductive effects (including congenital anomalies and low birth weight). There is some conflict in the literature investigating links between land contamination and cancers; the evidence for and against a link is equally balanced and is insufficient to make causal inferences. These are difficult to establish because of lack of data on individual exposures, and other socioeconomic and lifestyle factors that may confound a relationship with area of residence. There is no consistently occurring risk for any specific tumor across multiple studies on sites expected to contain similar contaminants. Further insights on health effects of land contamination are likely to be gained from studies that consider exposure pathways and biomarkers of exposure and effect, similar to those deployed with some success in investigating impacts of cadmium on human health.

  9. Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

  10. Evolutionary constraint and disease associations of post-translational modification sites in human genomes.

    Directory of Open Access Journals (Sweden)

    Jüri Reimand

    2015-01-01

    Full Text Available Interpreting the impact of human genome variation on phenotype is challenging. The functional effect of protein-coding variants is often predicted using sequence conservation and population frequency data, however other factors are likely relevant. We hypothesized that variants in protein post-translational modification (PTM sites contribute to phenotype variation and disease. We analyzed fraction of rare variants and non-synonymous to synonymous variant ratio (Ka/Ks in 7,500 human genomes and found a significant negative selection signal in PTM regions independent of six factors, including conservation, codon usage, and GC-content, that is widely distributed across tissue-specific genes and function classes. PTM regions are also enriched in known disease mutations, suggesting that PTM variation is more likely deleterious. PTM constraint also affects flanking sequence around modified residues and increases around clustered sites, indicating presence of functionally important short linear motifs. Using target site motifs of 124 kinases, we predict that at least ∼180,000 motif-breaker amino acid residues that disrupt PTM sites when substituted, and highlight kinase motifs that show specific negative selection and enrichment of disease mutations. We provide this dataset with corresponding hypothesized mechanisms as a community resource. As an example of our integrative approach, we propose that PTPN11 variants in Noonan syndrome aberrantly activate the protein by disrupting an uncharacterized cluster of phosphorylation sites. Further, as PTMs are molecular switches that are modulated by drugs, we study mutated binding sites of PTM enzymes in disease genes and define a drug-disease network containing 413 novel predicted disease-gene links.

  11. External human induced events in site evaluation for nuclear power plants. Safety guide

    International Nuclear Information System (INIS)

    2004-01-01

    The purpose of the present Safety Guide is to provide recommendations and guidance for the examination of the region considered for site evaluation for a plant in order to identity hazardous phenomena associated with human induced events initiated by sources external to the plant. In some cases it also presents preliminary guidance for deriving values of relevant parameters for the design basis. This Safety Guide is also applicable for periodic site evaluation and site evaluation following a major human induced event, and for the design and operation of the site's environmental monitoring system. Site evaluation includes site characterization. Consideration of external events that could lead to a degradation of the safety features of the plant and cause a release of radioactive material from the plant and/or affect the dispersion of such material in the environment. And consideration of population issues and access issues significant to safety (such as the feasibility of evacuation, the population distribution and the location of resources). The process of site evaluation continues throughout the lifetime of the facility, from siting to design, construction, operation and decommissioning. The external human induced events considered in this Safety Guide are all of accidental origin. Considerations relating to the physical protection of the plant against wilful actions by third parties are outside its scope. However, the methods described herein may also have some application for the purposes of such physical protection. The present Safety Guide may also be used for events that may originate within the boundaries of the site, but from sources which are not directly involved in the operational states of the nuclear power plant units, such as fuel depots or areas for the storage of hazardous materials for the construction of other facilities at the same site. Special consideration should be given to the hazardous material handled during the construction, operation and

  12. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

    Directory of Open Access Journals (Sweden)

    Misquitta Stephanie A

    2004-02-01

    Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

  13. Collagen cross-linking in sun-exposed and unexposed sites of aged human skin

    Science.gov (United States)

    Yamauchi, M.; Prisayanh, P.; Haque, Z.; Woodley, D. T.

    1991-01-01

    A recently described nonreducible, acid-heat stable compound, histidinohydroxylysinonorleucine (HHL), is a collagen cross-link isolated from mature skin tissue. Its abundance is related to chronologic aging of skin. The present communication describes the quantity of HHL from aged human skin of the same individuals in sun-exposed (wrist) and unexposed (buttock) sites. Punch biopsies were obtained from these sites from nine people of age 60 or older. HHL contents (moles/mole of collagen) at these sites were for wrist 0.13 +/- 0.07 and for buttock 0.69 +/- 0.17 (mean +/- SD, p less than 0.001). In addition, it was found that acute irradiation of the cross-linked peptides with UVA (up to 250 J/cm2) and UVB (up to 1 J/cm2) had no effect on HHL structure. The same treatment significantly degraded another nonreducible, stable collagen cross-link, pyridinoline. The results suggest that chronic sunlight exposure may be associated with an impediment to normal maturation of human dermal collagen resulting in tenuous amount of HHL. Thus, the process of photoaging in dermal collagen is different from that of chronologic aging in human skin.

  14. Ecological and human health sediment risk assessment for a hydrocarbon-impacted site in Lake Athabasca

    International Nuclear Information System (INIS)

    Mcdonald, B.; Wagenaar, A.; LaPorte, J.; Misfeldt, G.; Chatwell, I.

    2009-01-01

    The operation of a public port facility near Uranium City, Saskatchewan has resulted in elevated levels of hydrocarbons in soil, groundwater and sediment. Remedial action in the uplands portion of the site was successful and a risk management approach was initiated for the aquatic portion of the site in order to resolve human health and ecological issues. Ecological risks were assessed using a sediment weight-of-evidence approach involving chemistry, toxicity, bioaccumulation and benthic community structure. Human health risks were assessed via fish consumption, water ingestion and direct contact according to Health Canada guidance. This presentation included an overview of the general risk assessment approach as well as site-specific data and findings. The primary focus was on the challenges confronted during the risk assessment process, such as the need to include alkylated PAHs as a COPC in the human health risk assessment and to evaluate ongoing propeller wash and sediment resuspension for sediment risk management, even though the facility is no longer operational.

  15. Human cultural and related remains from Me Aure Cave (site WMD007), Moindou, New Caledonia

    International Nuclear Information System (INIS)

    Grant-Mackie, J.A.; Sand, C.; Valentin, F.; Fitzgerald, B.M.; Richer de Forges, B.

    2013-01-01

    In 1995 a small cave near Me Aure (site WMD007) on the west coast of New Caledonia, about 120 km northwest of Noumea, was excavated and found to contain mainly owl and human midden deposits. Some of the contents have already been documented and the present paper completes the study by reporting the human-related materials, including human bone fragments, pottery sherds, bones of four rodent species, and marine mollusc and crab remains. Each of these material classes are reported separately by the authors responsible for their analysis, and the results and interpretations based on each line of evidence are compared and contrasted. The human bone and pottery data suggest a temporally constrained deposit (2750-2350 BP) that has experienced stratigraphic disturbance. This result raises doubt about the un-mixed nature of the deposit emphasized in earlier publications and it urges instead the conclusion that the Me Aure stratigraphy consists mostly of a redeposited set of horizons. If this conclusion is correct, interpretations already published relying on a fixed chronology, especially about vegetation change and avifauna depletion or early aroid introduction will need to be reconsidered. The site constitutes the first in New Caledonia for which a cave deposit has now been fully analysed. (author). 36 refs., 11 figs., 9 tabs.

  16. Active site similarity between human and Plasmodium falciparum phosphodiesterases: considerations for antimalarial drug design

    Science.gov (United States)

    Howard, Brittany L.; Thompson, Philip E.; Manallack, David T.

    2011-08-01

    The similarity between Plasmodium falciparum phosphodiesterase enzymes ( PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

  17. Modelling the buried human body environment in upland climes using three contrasting field sites.

    Science.gov (United States)

    Wilson, Andrew S; Janaway, Robert C; Holland, Andrew D; Dodson, Hilary I; Baran, Eve; Pollard, A Mark; Tobin, Desmond J

    2007-06-14

    Despite an increasing literature on the decomposition of human remains, whether buried or exposed, it is important to recognise the role of specific microenvironments which can either trigger or delay the rate of decomposition. Recent casework in Northern England involving buried and partially buried human remains has demonstrated a need for a more detailed understanding of the effect of contrasting site conditions on cadaver decomposition and on the microenvironment created within the grave itself. Pigs (Sus scrofa) were used as body analogues in three inter-related taphonomy experiments to examine differential decomposition of buried human remains. They were buried at three contrasting field sites (pasture, moorland, and deciduous woodland) within a 15 km radius of the University of Bradford, West Yorkshire, UK. Changes to the buried body and the effect of these changes on hair and associated death-scene textile materials were monitored as was the microenvironment of the grave. At recovery, 6, 12 and 24 months post-burial, the extent of soft tissue decomposition was recorded and samples of fat and soil were collected for gas chromatography mass spectrometry (GCMS) analysis. The results of these studies demonstrated that (1) soil conditions at these three burial sites has a marked effect on the condition of the buried body but even within a single site variation can occur; (2) the process of soft tissue decomposition modifies the localised burial microenvironment in terms of microbiological load, pH, moisture and changes in redox status. These observations have widespread application for the investigation of clandestine burial and time since deposition, and in understanding changes within the burial microenvironment that may impact on biomaterials such as hair and other associated death scene materials.

  18. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  19. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  20. Visitors views of human origins after visiting the Cradle of Humankind World Heritage Site

    Directory of Open Access Journals (Sweden)

    Anthony Lelliott

    2016-02-01

    Full Text Available The Cradle of Humankind World Heritage Site, west of Johannesburg, was designated in 1999 because of its importance as a locality where numerous hominid fossils have been discovered since the 1930s. In this article, responses to questions from a survey of more than 800 adult visitors to the Cradle of Humankind visitor centres are analysed, covering their understanding of the concept of the "cradle" and their views on human evolution. Findings indicated that 63% of the respondents conceptualised the cradle as the origin or birthplace of humankind, and a similar proportion thought that nowhere else could be called the Cradle of Humankind (77% of people of South African nationality thought this. Nearly 60% of respondents accepted that humans evolved from an ape-like ancestor, while 25% disagreed. South Africans were less likely to accept human evolution than their international counterparts. The great majority of participants who accepted human evolution based their agreement on various forms of evidence and their knowledge of evolution. A religious foundation was used for their rationale by 60% of those who rejected evolution, with 33% citing evidence for their rejection. The implications of the findings are discussed in the light of public awareness and human origins.

  1. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  2. Fine resolution mapping of double-strand break sites for human ribosomal DNA units

    Directory of Open Access Journals (Sweden)

    Bernard J. Pope

    2016-12-01

    Full Text Available DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011 [5]; Blondet et al., 2001 Blondet et al. (2001 [1]. Stults et al. (2009 Stults et al. (2009 [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA restructuring is among the most common chromosomal alterations in adult solid tumours. As such, analysis of rDNA regions is likely to have significant prognostic and predictive value, clinically. Tchurikov et al. (2015a, 2016 Tchurikov et al. (2015a, 2016 [7,9] have made major advances in this direction, reporting that sites of human genome double-strand breaks (DSBs occur frequently at sites in rDNA that are tightly linked with active transcription - the authors used a RAFT (rapid amplification of forum termini protocol that selects for blunt-ended sites. They reported the relative frequency of these rDNA DSBs within defined co-ordinate ‘windows’ of varying size and made these data (as well as the relevant ‘raw’ sequencing information available to the public (Tchurikov et al., 2015b. Assay designs targeting rDNA DSB hotspots will benefit greatly from the publication of break sites at greater resolution. Here, we re-analyse public RAFT data and make available rDNA DSB co-ordinates to the single-nucleotide level.

  3. Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

    1985-03-18

    Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

  4. Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

    International Nuclear Information System (INIS)

    Mizuchi, A.; Iio, M.; Miyachi, Y.

    1984-01-01

    Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the β-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125 iodine. This assay was highly specific for HCG and there was no cross-reactivity with α,β-subunit of HCG, luteinizing hormone and follicle stimulating hormone. (Auth.)

  5. The genome of a Late Pleistocene human from a Clovis burial site in western Montana

    DEFF Research Database (Denmark)

    Rasmussen, Morten; Anzick, Sarah L.; Waters, Michael R

    2014-01-01

    Montana. The human bones date to 10,705 ± 35 (14)C years bp (approximately 12,707-12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal'ta population into Native...... directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western...

  6. Transferrin receptors on human reticulocytes: variation in site number in hematologic disorders

    International Nuclear Information System (INIS)

    Shumak, K.H.; Rachkewich, R.A.

    1984-01-01

    Assays of binding of 125iodine-labeled ( 125 I) human transferrin were used to study transferrin receptor sites on reticulocytes from 15 normal subjects and from 66 patients with various hematologic disorders. In normal subjects, few or no transferrin receptors were detected whereas the average number of receptors per reticulocyte varied greatly from patient to patient, ranging from 0 to 67,700 in samples, from 35 patients, on which Scatchard analysis of binding of [ 125 I]-transferrin was done. Marked heterogeneity in the number of reticulocyte transferrin receptors in different hematologic disorders was also found in assays with [ 125 I]-OKT9 (monoclonal antibody to the human transferrin receptor). The number of receptors was not correlated with either the reticulocyte count or the hemoglobin

  7. Handling of future human actions in the safety assessment SR-Site

    Energy Technology Data Exchange (ETDEWEB)

    2010-12-15

    This report documents the future human actions, FHA, considered in the long-term safety analysis of a KBS-3 repository. The report is one of the supporting documents to the safety assessment SR-Site (see further the Main report /SKB 2011/). The purpose of this report is to provide an account of general considerations concerning FHA, the methodology applied in SR-Site to assess FHA, the aspects of FHA needed to be considered in the evaluation of their impact on a deep geological repository and to select and analyse representative scenarios for illustrative consequence analysis. The main focus of this report is a time period when institutional control has ceased to be effective, thereby permitting inadvertent intrusion. However, a brief discussion of the earlier period when the repository has been closed, sealed and continuously kept under institutional control is also provided. General The potential exposure to large quantities of radiotoxic material is an inescapable consequence of the deposition of spent nuclear fuel in a final repository, and consequently intrusion into the repository needs to be considered in repository design and safety assessment. In accordance with ICRP recommendations /ICRP 2000/, intrusion in the post-closure phase of institutional control and beyond is primarily prevented through the design of the repository. In addition to that there will presumably continue to be safeguards measures, preservation of information (record keeping) and possibly some sort of markers placed at the site. During the institutional control period, activities at the site have to be restricted or directed if they have the potential to interfere with or hinder surveillance of the site, but this does not necessarily rule out all forms of access to the area. Also the fact that the repository contains fissile materials is an important aspect. Control of safeguards measures will most likely be upheld by national as well as international agencies. Furthermore, the

  8. Handling of future human actions in the safety assessment SR-Site

    International Nuclear Information System (INIS)

    2010-12-01

    This report documents the future human actions, FHA, considered in the long-term safety analysis of a KBS-3 repository. The report is one of the supporting documents to the safety assessment SR-Site (see further the Main report /SKB 2011/). The purpose of this report is to provide an account of general considerations concerning FHA, the methodology applied in SR-Site to assess FHA, the aspects of FHA needed to be considered in the evaluation of their impact on a deep geological repository and to select and analyse representative scenarios for illustrative consequence analysis. The main focus of this report is a time period when institutional control has ceased to be effective, thereby permitting inadvertent intrusion. However, a brief discussion of the earlier period when the repository has been closed, sealed and continuously kept under institutional control is also provided. General The potential exposure to large quantities of radiotoxic material is an inescapable consequence of the deposition of spent nuclear fuel in a final repository, and consequently intrusion into the repository needs to be considered in repository design and safety assessment. In accordance with ICRP recommendations /ICRP 2000/, intrusion in the post-closure phase of institutional control and beyond is primarily prevented through the design of the repository. In addition to that there will presumably continue to be safeguards measures, preservation of information (record keeping) and possibly some sort of markers placed at the site. During the institutional control period, activities at the site have to be restricted or directed if they have the potential to interfere with or hinder surveillance of the site, but this does not necessarily rule out all forms of access to the area. Also the fact that the repository contains fissile materials is an important aspect. Control of safeguards measures will most likely be upheld by national as well as international agencies. Furthermore, the

  9. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  10. Patterns of Snow Leopard Site Use in an Increasingly Human-Dominated Landscape.

    Directory of Open Access Journals (Sweden)

    Justine Shanti Alexander

    Full Text Available Human population growth and concomitant increases in demand for natural resources pose threats to many wildlife populations. The landscapes used by the endangered snow leopard (Panthera uncia and their prey is increasingly subject to major changes in land use. We aimed to assess the influence of 1 key human activities, as indicated by the presence of mining and livestock herding, and 2 the presence of a key prey species, the blue sheep (Pseudois nayaur, on probability of snow leopard site use across the landscape. In Gansu Province, China, we conducted sign surveys in 49 grid cells, each of 16 km2 in size, within a larger area of 3392 km2. We analysed the data using likelihood-based habitat occupancy models that explicitly account for imperfect detection and spatial auto-correlation between survey transect segments. The model-averaged estimate of snow leopard occupancy was high [0.75 (SE 0.10], but only marginally higher than the naïve estimate (0.67. Snow leopard segment-level probability of detection, given occupancy on a 500 m spatial replicate, was also high [0.68 (SE 0.08]. Prey presence was the main determinant of snow leopard site use, while human disturbances, in the form of mining and herding, had low predictive power. These findings suggest that snow leopards continue to use areas very close to such disturbances, as long as there is sufficient prey. Improved knowledge about the effect of human activity on large carnivores, which require large areas and intact prey populations, is urgently needed for conservation planning at the local and global levels. We highlight a number of methodological considerations that should guide the design of such research.

  11. Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site

    Directory of Open Access Journals (Sweden)

    Andrew R. Pepper

    2017-06-01

    Full Text Available Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of “healthy” human donor pancreata restricts the broader application of this effective curative therapy. “β-Like” cells derived from human embryonic stem cells (hESC, with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.

  12. Human-Specific Histone Methylation Signatures at Transcription Start Sites in Prefrontal Neurons

    Science.gov (United States)

    Cheung, Iris; Bharadwaj, Rahul; Chou, Hsin-Jung; Houston, Isaac B.; Peter, Cyril J.; Mitchell, Amanda C.; Yao, Wei-Dong; Myers, Richard H.; Chen, Jiang-fan; Preuss, Todd M.; Rogaev, Evgeny I.; Jensen, Jeffrey D.; Weng, Zhiping; Akbarian, Schahram

    2012-01-01

    Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5–1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans. PMID:23185133

  13. Impact of Alu repeats on the evolution of human p53 binding sites

    Directory of Open Access Journals (Sweden)

    Sirotin Michael V

    2011-01-01

    Full Text Available Abstract Background The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 in vivo binding sites (BSs reside in transposable repeats. The relationship between these BSs and functional p53 response elements (REs remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats. Results We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG → TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats. Conclusions The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu

  14. Human platelet ( sup 125 I)R-DOI binding sites. Characterization by in vitro autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Himeno, A.; Saavedra, J.M. (National Institute of Mental Health, Bethesda, MD (USA))

    1990-02-01

    We quantified binding sites for 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), a 5-HT2 agonist and hallucinogen, in human platelets. We incubated sections from human platelet pellets with ({sup 125}I)R-DOI with or without 1 mumol/L ketanserin, followed by autoradiography and computerized microdensitometry. We corrected the values of binding density by the protein content of each section with a densitometric protein assay. The present method revealed a single class of high affinity binding sites for ({sup 125}I)R-DOI, with a Kd of 6.4 +/- 0.7 nmol/L and a Bmax of 100 +/- 10 fmol/mg protein. Kd and Bmax for ({sup 125}I)R-DOI determined by the classical membrane binding assay, were 2.7 +/- 0.4 nmol/L and 100 +/- 10 fmol/mg protein, respectively. The present method is precise, very sensitive, and allows the characterization of ({sup 125}I)R-DOI binding in sections obtained from as little as 3 ml of blood. Standardization is possible after correction by the protein content of each individual section.

  15. Recruitment of host's progenitor cells to sites of human amniotic fluid stem cells implantation.

    Science.gov (United States)

    Mirabella, Teodelinda; Poggi, Alessandro; Scaranari, Monica; Mogni, Massimo; Lituania, Mario; Baldo, Chiara; Cancedda, Ranieri; Gentili, Chiara

    2011-06-01

    The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Study of arsenic contents in human hair of contrast sites in Bangladesh

    International Nuclear Information System (INIS)

    Hafiz, M.A.; Hossain, S.M.; Arafat, Y.

    2007-01-01

    Arsenic concentrations in human hair samples of a highly polluted site namely Boro Dudpalila village, Damurhuda, Chuadanga and nonpolluted sites of Goainghat and Sylhet Sadar thanas were determined using instrumental neutron activation analysis (INAA) technique. Samples were irradiated in the TRIGA Mark-II research reactor of Atomic Energy Research Establishment (AERE), Savar, Dhaka, Bangladesh and PARR-2 of Pakistan Institute of Nuclear Science and Technology (PINSTECH), Islamabad, Pakistan at a thermal neutron flux of order 10 12 n/cm 2 /s for 3 hours. Decay time was about 2 days. Measurement time was 2700 sec for Dhaka and 1800 sec for Islamabad laboratories. HPGe detectors were used for γ-ray measurement. Ranges of arsenic concentrations in Chuadanga and Sylhet samples were found to be 1.04±0.06 to 48.66±1.32 and <0.20 to 0.84±0.04 ppm, respectively. Minimum detection limit of arsenic in the hair samples was found to be 0.20 ppm. All Chuadanga samples exceeded the normal level of arsenic in human hair (1 ppm). In the study it was found that both males and females are affected and there was generally no consistency in the arsenic levels in hair of the members of the same family. (author)

  17. Competitive binding of Chlorin p6 and Dansyl-L-Proline to Sudlow's site II of human serum albumin

    Science.gov (United States)

    Patel, Sunita; Sharma, Kaushal Kishor; Datta, Anindya

    2015-03-01

    The binding of chlorin p6, a model photosensitizer for photodynamic therapy (PDT), to the Sudlow's site II of Human Serum Albumin (HSA) has been monitored by different spectroscopic methods. Displacement of Dansyl-L-Proline (DP) from its conjugate with HSA is manifested in the spectral shift and decrease in its fluorescence intensity as well as the emergence of component with lifetime of 2-3 ns, which is characteristic of free DP. As DP is known to bind specifically to the Sudlow's site II of human serum albumin, its displacement by chlorin p6 indicates the residence of the photosensitizer in the same site, in addition to Sudlow's site I. The binding constants for Sudlow's site II, determined by the stopped-flow technique, are found to be two orders of magnitude smaller than that for Sudlow's site I.

  18. Communication across 300 generations: deterring human interference with waste deposit sites

    International Nuclear Information System (INIS)

    Tannenbaum, P.H.

    1984-04-01

    The conditions attendant on the deep land burial of nuclear waste products raise a number of possible scenarios to cover the necessary 10,000 years of burial. However, no matter what kind of futuristic scenario obtains, it is desirable to develop an information system indicating the locale and nature of the deposit site and the types of materials stored, along with forewarnings not to interefere with the sites. A variety of such informational sites are suggested. Attention then turns to the recipients of such messages, recognizing from the outset that the psychological/perceptual makeup of individuals across the next 300 or so generations is virtually impossible to predict, particularly since new technologies may well alter that makeup in the furture. Nevertheless, current evidence suggests that certain human characteristics may be considered universal, and that these suggest the incorporation of selected sign signification into the message system. There are other such characteristics that, while probably not intrinsic, can probably be acquired with a minimum of formal training. That still leaves much of the message content to be deliberately created and, hence, learned. The common trefoil or other developed biohazardous signs emerge as the best candidates for a generic base symbol for the buried material

  19. Non-site-specific allosteric effect of oxygen on human hemoglobin under high oxygen partial pressure.

    Science.gov (United States)

    Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

    2014-04-08

    Protein allostery is essential for vital activities. Allosteric regulation of human hemoglobin (HbA) with two quaternary states T and R has been a paradigm of allosteric structural regulation of proteins. It is widely accepted that oxygen molecules (O2) act as a "site-specific" homotropic effector, or the successive O2 binding to the heme brings about the quaternary regulation. However, here we show that the site-specific allosteric effect is not necessarily only a unique mechanism of O2 allostery. Our simulation results revealed that the solution environment of high O2 partial pressure enhances the quaternary change from T to R without binding to the heme, suggesting an additional "non-site-specific" allosteric effect of O2. The latter effect should play a complementary role in the quaternary change by affecting the intersubunit contacts. This analysis must become a milestone in comprehensive understanding of the allosteric regulation of HbA from the molecular point of view.

  20. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  1. Communication across 300 generations: deterring human interference with waste deposit sites

    Energy Technology Data Exchange (ETDEWEB)

    Tannenbaum, P.H.

    1984-04-01

    The conditions attendant on the deep land burial of nuclear waste products raise a number of possible scenarios to cover the necessary 10,000 years of burial. However, no matter what kind of futuristic scenario obtains, it is desirable to develop an information system indicating the locale and nature of the deposit site and the types of materials stored, along with forewarnings not to interefere with the sites. A variety of such informational sites are suggested. Attention then turns to the recipients of such messages, recognizing from the outset that the psychological/perceptual makeup of individuals across the next 300 or so generations is virtually impossible to predict, particularly since new technologies may well alter that makeup in the furture. Nevertheless, current evidence suggests that certain human characteristics may be considered universal, and that these suggest the incorporation of selected sign signification into the message system. There are other such characteristics that, while probably not intrinsic, can probably be acquired with a minimum of formal training. That still leaves much of the message content to be deliberately created and, hence, learned. The common trefoil or other developed biohazardous signs emerge as the best candidates for a generic base symbol for the buried material.

  2. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen

    International Nuclear Information System (INIS)

    Lantz, M.S.; Allen, R.D.; Bounelis, P.; Switalski, L.M.; Hook, M.

    1990-01-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains

  3. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Lantz, M.S.; Allen, R.D.; Bounelis, P.; Switalski, L.M.; Hook, M. (Univ. of Alabama, Birmingham (USA))

    1990-02-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.

  4. Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

    International Nuclear Information System (INIS)

    Klimov, Eugene; Vinokourova, Svetlana; Moisjak, Elena; Rakhmanaliev, Elian; Kobseva, Vera; Laimins, Laimonis; Kisseljov, Fjodor; Sulimova, Galina

    2002-01-01

    In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA

  5. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Directory of Open Access Journals (Sweden)

    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  6. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    Science.gov (United States)

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  7. Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors.

    Science.gov (United States)

    Schmitz, Janina; Furtmann, Norbert; Ponert, Moritz; Frizler, Maxim; Löser, Reik; Bartz, Ulrike; Bajorath, Jürgen; Gütschow, Michael

    2015-08-01

    Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A GIS-based approach for the long-term prediction of human health risks at contaminated sites

    NARCIS (Netherlands)

    Bień, J.D.; Meer, J. ter; Rulkens, W.H.; Rijnaarts, H.H.M.

    2005-01-01

    A Health Index/Risk Evaluation Tool (HIRET) has been developed for the integration of risk assessment and spatial planning using GIS capabilities. The method is meant to assist decision makers and site owners in the evaluation of potential human health risk with respect to land use. Human health

  9. A GIS-based approach for the long-term prediction of human health risks at contaminated sites

    NARCIS (Netherlands)

    Bien, J.D.; Meer, J.; Rulkens, W.H.; Rijnaarts, H.H.M.

    2004-01-01

    A Health Index/Risk Evaluation Tool (HIRET) has been developed for the integration of risk assessment and spatial planning using GIS capabilities. The method is meant to assist decision makers and site owners in the evaluation of potential human health risk with respect to land use. Human health

  10. Morphology of muscle attachment sites in the modern human hand does not reflect muscle architecture.

    Science.gov (United States)

    Williams-Hatala, E M; Hatala, K G; Hiles, S; Rabey, K N

    2016-06-23

    Muscle attachment sites (entheses) on dry bones are regularly used by paleontologists to infer soft tissue anatomy and to reconstruct behaviors of extinct organisms. This method is commonly applied to fossil hominin hand bones to assess their abilities to participate in Paleolithic stone tool behaviors. Little is known, however, about how or even whether muscle anatomy and activity regimes influence the morphologies of their entheses, especially in the hand. Using the opponens muscles from a sample of modern humans, we tested the hypothesis that aspects of hand muscle architecture that are known to be influenced by behavior correlate with the size and shape of their associated entheses. Results show no consistent relationships between these behaviorally-influenced aspects of muscle architecture and entheseal morphology. Consequently, it is likely premature to infer patterns of behavior, such as stone tool making in fossil hominins, from these same entheses.

  11. Site specific mineral composition and microstructure of human supra-gingival dental calculus.

    Science.gov (United States)

    Hayashizaki, Junko; Ban, Seiji; Nakagaki, Haruo; Okumura, Akihiko; Yoshii, Saori; Robinson, Colin

    2008-02-01

    Dental calculus has been implicated in the aetiology of several periodontal conditions. Its prevention and removal are therefore desirable clinical goals. While it is known that calculus is very variable in chemical composition, crystallinity and crystallite size little is known about site specific variability within a dentition and between individuals. With this in mind, a study was undertaken to investigate the comparative site specific nature and composition of human dental supra-gingival dental calculus obtained from 66 male patients visiting for their dental check-up using fluorescent X-ray spectroscopy, X-ray diffractometry and Fourier transform infrared spectroscopy. The supra-gingival dental calculus formed on the lingual surfaces of lower anterior teeth and the buccal surfaces of upper molar teeth were classified into four types based on calcium phosphate phases present. There was significant difference in composition of the crystal phase types between lower and upper teeth (pdental calculus on anterior or molar teeth of all samples. The degree of crystallinity of dental calculus formed on the upper molar teeth was higher than that formed on the lower anterior teeth (pdental calculus formed on the lower anterior teeth were higher than on upper molar teeth (pdental supra-gingival dental calculus is related to its location in the mouth.

  12. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    International Nuclear Information System (INIS)

    Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

    2006-01-01

    The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å

  13. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    Science.gov (United States)

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  14. Body Site Is a More Determinant Factor than Human Population Diversity in the Healthy Skin Microbiome.

    Directory of Open Access Journals (Sweden)

    Guillermo I Perez Perez

    Full Text Available We studied skin microbiota present in three skin sites (forearm, axilla, scalp in men from six ethnic groups living in New York City.Samples were obtained at baseline and after four days following use of neutral soap and stopping regular hygiene products, including shampoos and deodorants. DNA was extracted using the MoBio Power Lyzer kit and 16S rRNA gene sequences determined on the IIlumina MiSeq platform, using QIIME for analysis.Our analysis confirmed skin swabbing as a useful method for sampling different areas of the skin because DNA concentrations and number of sequences obtained across subject libraries were similar. We confirmed that skin location was the main factor determining the composition of bacterial communities. Alpha diversity, expressed as number of species observed, was greater in arm than on scalp or axilla in all studied groups. We observed an unexpected increase in α-diversity on arm, with similar tendency on scalp, in the South Asian group after subjects stopped using their regular shampoos and deodorants. Significant differences at phylum and genus levels were observed between subjects of the different ethnic origins at all skin sites.We conclude that ethnicity and particular soap and shampoo practices are secondary factors compared to the ecological zone of the human body in determining cutaneous microbiota composition.

  15. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    International Nuclear Information System (INIS)

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G.

    2006-01-01

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (ΔUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the ΔUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or ΔUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites

  16. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Hao [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Yu, Wen-bo [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Li, Shu-xing [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Ding, Xiang-ming; Yu, Long [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Bi, Ru-Chang [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2006-02-01

    The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å.

  17. High DNA melting temperature predicts transcription start site location in human and mouse.

    LENUS (Irish Health Repository)

    Dineen, David G

    2009-12-01

    The accurate computational prediction of transcription start sites (TSS) in vertebrate genomes is a difficult problem. The physicochemical properties of DNA can be computed in various ways and a many combinations of DNA features have been tested in the past for use as predictors of transcription. We looked in detail at melting temperature, which measures the temperature, at which two strands of DNA separate, considering the cooperative nature of this process. We find that peaks in melting temperature correspond closely to experimentally determined transcription start sites in human and mouse chromosomes. Using melting temperature alone, and with simple thresholding, we can predict TSS with accuracy that is competitive with the most accurate state-of-the-art TSS prediction methods. Accuracy is measured using both experimentally and manually determined TSS. The method works especially well with CpG island containing promoters, but also works when CpG islands are absent. This result is clear evidence of the important role of the physical properties of DNA in the process of transcription. It also points to the importance for TSS prediction methods to include melting temperature as prior information.

  18. Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

    Science.gov (United States)

    Qiu, Huawei; Boudanova, Ekaterina; Park, Anna; Bird, Julie J; Honey, Denise M; Zarazinski, Christine; Greene, Ben; Kingsbury, Jonathan S; Boucher, Susan; Pollock, Julie; McPherson, John M; Pan, Clark Q

    2013-03-20

    Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and β subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

  19. Divergent evolution of human p53 binding sites: cell cycle versus apoptosis.

    Directory of Open Access Journals (Sweden)

    Monica M Horvath

    2007-07-01

    Full Text Available The p53 tumor suppressor is a sequence-specific pleiotropic transcription factor that coordinates cellular responses to DNA damage and stress, initiating cell-cycle arrest or triggering apoptosis. Although the human p53 binding site sequence (or response element [RE] is well characterized, some genes have consensus-poor REs that are nevertheless both necessary and sufficient for transactivation by p53. Identification of new functional gene regulatory elements under these conditions is problematic, and evolutionary conservation is often employed. We evaluated the comparative genomics approach for assessing evolutionary conservation of putative binding sites by examining conservation of 83 experimentally validated human p53 REs against mouse, rat, rabbit, and dog genomes and detected pronounced conservation differences among p53 REs and p53-regulated pathways. Bona fide NRF2 (nuclear factor [erythroid-derived 2]-like 2 nuclear factor and NFkappaB (nuclear factor of kappa light chain gene enhancer in B cells binding sites, which direct oxidative stress and innate immunity responses, were used as controls, and both exhibited high interspecific conservation. Surprisingly, the average p53 RE was not significantly more conserved than background genomic sequence, and p53 REs in apoptosis genes as a group showed very little conservation. The common bioinformatics practice of filtering RE predictions by 80% rodent sequence identity would not only give a false positive rate of approximately 19%, but miss up to 57% of true p53 REs. Examination of interspecific DNA base substitutions as a function of position in the p53 consensus sequence reveals an unexpected excess of diversity in apoptosis-regulating REs versus cell-cycle controlling REs (rodent comparisons: p < 1.0 e-12. While some p53 REs show relatively high levels of conservation, REs in many genes such as BAX, FAS, PCNA, CASP6, SIVA1, and P53AIP1 show little if any homology to rodent sequences. This

  20. The first trimester human placenta is a site for terminal maturation of primitive erythroid cells.

    Science.gov (United States)

    Van Handel, Ben; Prashad, Sacha L; Hassanzadeh-Kiabi, Nargess; Huang, Andy; Magnusson, Mattias; Atanassova, Boriana; Chen, Angela; Hamalainen, Eija I; Mikkola, Hanna K A

    2010-10-28

    Embryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryo's immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in first trimester placental villi. Extravascular ζ-globin(+) primitive erythroid cells were found in placental villi between 5-7 weeks of development, at which time the frequency of enucleated RBCs was higher in the villous stroma than in circulation. RBC enucleation was further evidenced by the presence of primitive reticulocytes and pyrenocytes (ejected RBC nuclei) in the placenta. Extravascular RBCs were found to associate with placental macrophages, which contained ingested nuclei. Clonogenic macrophage progenitors of fetal origin were present in the chorionic plate of the placenta before the onset of fetoplacental circulation, after which macrophages had migrated to the villi. These findings indicate that placental macrophages may assist the enucleation process of primitive RBCs in placental villi, implying an unexpectedly broad role for the placenta in embryonic hematopoiesis.

  1. Integration of HIV in the Human Genome: Which Sites Are Preferential? A Genetic and Statistical Assessment

    Science.gov (United States)

    Gonçalves, Juliana; Moreira, Elsa; Sequeira, Inês J.; Rodrigues, António S.; Rueff, José; Brás, Aldina

    2016-01-01

    Chromosomal fragile sites (FSs) are loci where gaps and breaks may occur and are preferential integration targets for some viruses, for example, Hepatitis B, Epstein-Barr virus, HPV16, HPV18, and MLV vectors. However, the integration of the human immunodeficiency virus (HIV) in Giemsa bands and in FSs is not yet completely clear. This study aimed to assess the integration preferences of HIV in FSs and in Giemsa bands using an in silico study. HIV integration positions from Jurkat cells were used and two nonparametric tests were applied to compare HIV integration in dark versus light bands and in FS versus non-FS (NFSs). The results show that light bands are preferential targets for integration of HIV-1 in Jurkat cells and also that it integrates with equal intensity in FSs and in NFSs. The data indicates that HIV displays different preferences for FSs compared to other viruses. The aim was to develop and apply an approach to predict the conditions and constraints of HIV insertion in the human genome which seems to adequately complement empirical data. PMID:27294106

  2. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Science.gov (United States)

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Rijken, D.C.; Juhan-Vague, I.; De Cock, F.; Collen, D.

    1983-01-01

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 μl containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125 I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator

  4. Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

    Science.gov (United States)

    DeVry, C G; Tsai, W; Clarke, S

    1996-11-15

    The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

  5. Standardized UXO Technology Demonstration Site Open Field Scoring Recording Number 231 (Human Factors Applications, Inc.)

    National Research Council Canada - National Science Library

    Overbay, Larry; Robitaille, George

    2005-01-01

    ...) utilizing the APG Standardized UXO Technology Demonstration Site Open Field. The scoring record was coordinated by Larry Overbuy and by the Standardized UXO Technology Demonstration Site Scoring Committee...

  6. Shared regulatory sites are abundant in the human genome and shed light on genome evolution and disease pleiotropy.

    Science.gov (United States)

    Tong, Pin; Monahan, Jack; Prendergast, James G D

    2017-03-01

    Large-scale gene expression datasets are providing an increasing understanding of the location of cis-eQTLs in the human genome and their role in disease. However, little is currently known regarding the extent of regulatory site-sharing between genes. This is despite it having potentially wide-ranging implications, from the determination of the way in which genetic variants may shape multiple phenotypes to the understanding of the evolution of human gene order. By first identifying the location of non-redundant cis-eQTLs, we show that regulatory site-sharing is a relatively common phenomenon in the human genome, with over 10% of non-redundant regulatory variants linked to the expression of multiple nearby genes. We show that these shared, local regulatory sites are linked to high levels of chromatin looping between the regulatory sites and their associated genes. In addition, these co-regulated gene modules are found to be strongly conserved across mammalian species, suggesting that shared regulatory sites have played an important role in shaping human gene order. The association of these shared cis-eQTLs with multiple genes means they also appear to be unusually important in understanding the genetics of human phenotypes and pleiotropy, with shared regulatory sites more often linked to multiple human phenotypes than other regulatory variants. This study shows that regulatory site-sharing is likely an underappreciated aspect of gene regulation and has important implications for the understanding of various biological phenomena, including how the two and three dimensional structures of the genome have been shaped and the potential causes of disease pleiotropy outside coding regions.

  7. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  8. Genome-Wide Analysis of PAPS1-Dependent Polyadenylation Identifies Novel Roles for Functionally Specialized Poly(A Polymerases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Christian Kappel

    2015-08-01

    Full Text Available The poly(A tail at 3' ends of eukaryotic mRNAs promotes their nuclear export, stability and translational efficiency, and changes in its length can strongly impact gene expression. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A polymerases, PAPS1, PAPS2 and PAPS4. As shown by their different mutant phenotypes, these three isoforms are functionally specialized, with PAPS1 modifying organ growth and suppressing a constitutive immune response. However, the molecular basis of this specialization is largely unknown. Here, we have estimated poly(A-tail lengths on a transcriptome-wide scale in wild-type and paps1 mutants. This identified categories of genes as particularly strongly affected in paps1 mutants, including genes encoding ribosomal proteins, cell-division factors and major carbohydrate-metabolic proteins. We experimentally verified two novel functions of PAPS1 in ribosome biogenesis and redox homoeostasis that were predicted based on the analysis of poly(A-tail length changes in paps1 mutants. When overlaying the PAPS1-dependent effects observed here with coexpression analysis based on independent microarray data, the two clusters of transcripts that are most closely coexpressed with PAPS1 show the strongest change in poly(A-tail length and transcript abundance in paps1 mutants in our analysis. This suggests that their coexpression reflects at least partly the preferential polyadenylation of these transcripts by PAPS1 versus the other two poly(A-polymerase isoforms. Thus, transcriptome-wide analysis of poly(A-tail lengths identifies novel biological functions and likely target transcripts for polyadenylation by PAPS1. Data integration with large-scale co-expression data suggests that changes in the relative activities of the isoforms are used as an endogenous mechanism to co-ordinately modulate plant gene expression.

  9. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  10. On-site preparation of technetium-99m labeled human serum albumin for clinical application

    International Nuclear Information System (INIS)

    Wang Yuhfeng; Chuang Meihua; Cham Thauming; Chung Meiing; Chiu Jainnshiun

    2007-01-01

    Technetium-99m labeled human serum albumin (Tc-99m HSA) is an important radiopharmaceutical for clinical applications, such as cardiac function tests or protein-losing gastroenteropathy assessment. However, because of transfusion-induced infectious diseases, the safety of serum products is a serious concern. In this context, serum products acquired from patients themselves are the most ideal tracer. However, the development of rapid separation and easy clinical labeling methods is not yet well established. Under such situation, products from the same ethnic group or country are now recommended by the World Health Organization as an alternative preparation. This article describes the on-site preparation of Tc-99m HSA from locally supplied serum products. Different formulations were prepared and the labeling efficiency and stability were examined. Radio-labeling efficiencies were more than 90% in all preparation protocols, except for one that omitted the stannous solution. The most cost-effective protocol contained HSA 0.1 mg, treated with stannous fluoride 0.2 mg, and mixed with Tc-99m pertechnetate 30 mCi. A biodistribution study was performed in rats using a gamma camera immediately after intravenous administration of radiolabeled HSA. Tissue/organ uptake was obtained by measuring the radioactivity in organs after sacrificing the rats at timed intervals. The biologic half-life was about 32 min, determined from sequential venous blood collections. These data indicate that our preparation of Tc-99m HSA is useful and potentially applicable clinically. In addition, this on-site preparation provides the possibility of labeling a patient's own serum for subsequent clinical application. (author)

  11. Direct measurement of human plasma corticotropin-releasing hormone by two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Linton, E.A.; McLean, C.; Nieuwenhuyzen Kruseman, A.C.; Tilders, F.J.; Van der Veen, E.A.; Lowry, P.J.

    1987-01-01

    A ''two-site'' immunoradiometric assay (IRMA) which allows the direct estimation of human CRH (hCRH) in plasma is described. Using this IRMA, basal levels of CRH in normal subjects ranged from 2-28 pg/mL [mean, 15 +/- 7 (+/- SD) pg/mL; n = 58]. Values in men and women were similar. Plasma CRH values within this range were also found in patients with Cushing's syndrome, Addison's disease, and Nelson's syndrome, with no correlation between plasma CRH and ACTH levels in these patients. Elevated plasma CRH levels were found in pregnant women near term [1462 +/- 752 (+/- SD) pg/mL; n = 55], and the dilution curve of this CRH-like immunoreactivity paralleled the IRMA standard curve. After its immunoadsorption from maternal plasma, this CRH-like material eluted on reverse phase high performance liquid chromatography with a retention time identical to that of synthetic CRH and had equipotent bioactivity with the synthetic peptide in the perfused anterior pituitary cell bioassay. Circulating CRH was not detected in Wistar rats, even after adrenalectomy and subsequent ether stress. Synthetic hCRH was degraded by fresh human plasma relatively slowly; 65% of added CRH remained after 1 h of incubation at 37 C. Degradation was inhibited by heat treatment (54 C; 1 h), cold treatment (4 C; 4 h), or freezing and thawing. Loss of synthetic rat CRH occurred more rapidly when fresh rat plasma was used; only 20% of added CRH remained under the same conditions. The inability to measure CRH in peripheral rat plasma may be due to the presence of active CRH-degrading enzymes which fragment the CRH molecule into forms not recognized by the CRH IRMA

  12. Human β-globin locus control region: Analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice

    International Nuclear Information System (INIS)

    Caterina, J.J.; Ryan, T.M.; Pawlik, K.M.; Townes, T.M.; Brinster, R.L.; Behringer, R.R.; Palmiter, R.D.

    1991-01-01

    The human β-globin locus control region (LCR) is essential for high-level expression of human var-epsilon-, γ-, and β-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human β-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human β-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletion of both 5' and 3' sequences, a 373-base-pair (BP) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 19-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained

  13. The early noncoding region of human papillomavirus type 16 is regulated by cytoplasmic polyadenylation factors

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Kristiansen, Karen; Durand, Marjorie

    2010-01-01

    All human papillomavirus type 16 (HPV-16) early mRNAs are polyadenylated at the poly(A) signal within the early 3' untranslated region (3'UTR). The 3'end of the early E5 open reading frame and the 3'UTR of HPV-16 is very AU-rich, with five regions similar to cytoplasmic polyadenylation elements (...

  14. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology.

    Science.gov (United States)

    Liu, Ying; Lu, Zheming; Xu, Ruiping; Ke, Yang

    2016-02-02

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma.

  15. Characterization of site-specific biomechanical properties of human meniscus-Importance of collagen and fluid on mechanical nonlinearities.

    Science.gov (United States)

    Danso, E K; Mäkelä, J T A; Tanska, P; Mononen, M E; Honkanen, J T J; Jurvelin, J S; Töyräs, J; Julkunen, P; Korhonen, R K

    2015-06-01

    Meniscus adapts to joint loads by depth- and site-specific variations in its composition and structure. However, site-specific mechanical characteristics of intact meniscus under compression are poorly known. In particular, mechanical nonlinearities caused by different meniscal constituents (collagen and fluid) are not known. In the current study, in situ indentation testing was conducted to determine site-specific elastic, viscoelastic and poroelastic properties of intact human menisci. Lateral and medial menisci (n=26) were harvested from the left knee joint of 13 human cadavers. Indentation tests, using stress-relaxation and dynamic (sinusoidal) loading protocols, were conducted for menisci at different sites (anterior, middle, posterior, n=78). Sample- and site-specific axisymmetric finite element models with fibril-reinforced poroelastic properties were fitted to the corresponding stress-relaxation curves to determine the mechanical parameters. Elastic moduli, especially the instantaneous and dynamic moduli, showed site-specific variation only in the medial meniscus (pmeniscus. The phase angle showed no statistically significant variation between the sites (p>0.05). The values for the strain-dependent fibril network modulus (nonlinear behaviour of collagen) were significantly different (pmeniscus only between the middle and posterior sites. For the strain-dependent permeability coefficient, only anterior and middle sites showed a significant difference (pmeniscus. This parameter demonstrated a significant difference (pmeniscus shows more site-dependent variation in the mechanical properties as compared to lateral meniscus. In particular, anterior horn of medial meniscus was the stiffest and showed the most nonlinear mechanical behaviour. The nonlinearity was related to both collagen fibrils and fluid. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Passive Barriers to Inadvertent Human Intrusion for Use at the Nevada Test Site

    International Nuclear Information System (INIS)

    NSTec Environmental Management

    2007-01-01

    In July1996, BN transmitted Passive Barriers to Inadvertent Human Intrusion for Use at the Nevada Test Site to the United States Department of Energy, under Contract DE-AC08-91NV10833. The 1996 paper had a limited distribution and was not reviewed for public release. In 2007, National Security Technologies LLC (NSTec) made minor revisions to conform to current editorial standards of the NNSA/NSO and to meet current security requirements for public release. The primary purpose of this study was to identify types of engineered passive barriers that could deter future intrusion into buried low-level radioactive waste, particularly intrusion by drilling water wells. The study considered drilling technology, many natural and man-made materials, and both underground and above-ground barriers. Based on cost and effectiveness, the report recommended underground barriers consisting of a layer of rubble or tires. An aboveground barrier mound might also prove effective, but would cost more, and may become an attractive nuisance (e.g., might, after their purpose has been forgotten, encourage exploration for the sake of satisfying curiosity). Advances in drilling technology could render any engineered barriers ineffective if there is motivation to penetrate the barriers

  17. HOCOMOCO: a comprehensive collection of human transcription factor binding sites models

    Science.gov (United States)

    Kulakovskiy, Ivan V.; Medvedeva, Yulia A.; Schaefer, Ulf; Kasianov, Artem S.; Vorontsov, Ilya E.; Bajic, Vladimir B.; Makeev, Vsevolod J.

    2013-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. PMID:23175603

  18. Delineation of the peptide binding site of the human galanin receptor.

    Science.gov (United States)

    Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

    1996-01-01

    Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

  19. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.; Medvedeva, Yulia A.; Schaefer, Ulf; Kasianov, Artem S.; Vorontsov, Ilya E.; Bajic, Vladimir B.; Makeev, Vsevolod J.

    2012-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  20. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  1. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot....... In the simulations, the cavities are completely filled with water molecules, although with approximately 20% lower density than in bulk water. The 2A6 protein differs from the other three in that it has a very small cavity with only two water molecules and no exchange with the surroundings. The other three proteins...... channels, through which there is a quite frequent exchange of water molecules (one molecule is exchanged every 30-200 ps), except in 2A6. Most of the channels are observed also in the crystal structures, but two to three channels in each protein open only during the simulations. There are no water...

  2. 5' diversity of human hepatic PXR (NR1I2) transcripts and identification of the major transcription initiation site.

    Science.gov (United States)

    Kurose, Kouichi; Koyano, Satoru; Ikeda, Shinobu; Tohkin, Masahiro; Hasegawa, Ryuichi; Sawada, Jun-Ichi

    2005-05-01

    The human pregnane X receptor (PXR) is a crucial regulator of the genes encoding several major cytochrome P450 enzymes and transporters, such as CYP3A4 and MDR1, but its own transcriptional regulation remains unclear. To elucidate the transcriptional mechanisms of human PXR gene, we first endeavored to identify the transcription initiation site of human PXR using 5'-RACE. Five types of 5'-variable transcripts (a, b, c, d, and e) with common exon 2 sequence were found, and comparison of these sequences with the genomic sequence suggested that their 5' diversity is derived from initiation by alternative promoters and alternative splicing. None of the exons found in our study contain any new in-frame coding regions. Newly identified introns IVS-a and IVS-b were found to have CT-AC splice sites that do not follow the GT-AG rule of conventional donor and acceptor splice sites. Of the five types of 5' variable transcripts identified, RT-PCR showed that type-a was the major transcript type. Four transcription initiation sites (A-D) for type-a transcript were identified by 5'-RACE using GeneRacer RACE Ready cDNA (human liver) constructed by the oligo-capping method. Putative TATA boxes were located approximately 30 bp upstream from the transcriptional start sites of the major transcript (C) and the longest minor transcript (A) expressed in the human liver. These results indicate that the initiation of transcription of human PXR is more complex than previously reported.

  3. New polymorphic variants of human blood clotting factor IX

    Energy Technology Data Exchange (ETDEWEB)

    Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V. [Hematological Research Center, Moscow (Russian Federation); Plutalov, O.V.; Berlin, Yu.A. [Shemyakin Institute of Bioorganic Chemistry, Moscow (Russian Federation)

    1995-04-01

    The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

  4. Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

    International Nuclear Information System (INIS)

    Mak, J.C.; Barnes, P.J.

    1988-01-01

    125 I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125 I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow

  5. Homo sapiens-Specific Binding Site Variants within Brain Exclusive Enhancers Are Subject to Accelerated Divergence across Human Population.

    Science.gov (United States)

    Zehra, Rabail; Abbasi, Amir Ali

    2018-03-01

    Empirical assessments of human accelerated noncoding DNA frgaments have delineated presence of many cis-regulatory elements. Enhancers make up an important category of such accelerated cis-regulatory elements that efficiently control the spatiotemporal expression of many developmental genes. Establishing plausible reasons for accelerated enhancer sequence divergence in Homo sapiens has been termed significant in various previously published studies. This acceleration by including closely related primates and archaic human data has the potential to open up evolutionary avenues for deducing present-day brain structure. This study relied on empirically confirmed brain exclusive enhancers to avoid any misjudgments about their regulatory status and categorized among them a subset of enhancers with an exceptionally accelerated rate of lineage specific divergence in humans. In this assorted set, 13 distinct transcription factor binding sites were located that possessed unique existence in humans. Three of 13 such sites belonging to transcription factors SOX2, RUNX1/3, and FOS/JUND possessed single nucleotide variants that made them unique to H. sapiens upon comparisons with Neandertal and Denisovan orthologous sequences. These variants modifying the binding sites in modern human lineage were further substantiated as single nucleotide polymorphisms via exploiting 1000 Genomes Project Phase3 data. Long range haplotype based tests laid out evidence of positive selection to be governing in African population on two of the modern human motif modifying alleles with strongest results for SOX2 binding site. In sum, our study acknowledges acceleration in noncoding regulatory landscape of the genome and highlights functional parts within it to have undergone accelerated divergence in present-day human population.

  6. Evaluating Potential Human Health Risks Associated with the Development of Utility-Scale Solar Energy Facilities on Contaminated Sites

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, J. -J. [Argonne National Lab. (ANL), Argonne, IL (United States); Chang, Y. -S. [Argonne National Lab. (ANL), Argonne, IL (United States); Hartmann, H. [Argonne National Lab. (ANL), Argonne, IL (United States); Wescott, K. [Argonne National Lab. (ANL), Argonne, IL (United States); Kygeris, C. [Indiana Univ. of Pennsylvania, PA (United States)

    2013-09-01

    This report presents a general methodology for obtaining preliminary estimates of the potential human health risks associated with developing a utility-scale solar energy facility on a contaminated site, based on potential exposures to contaminants in soils (including transport of those contaminants into the air).

  7. A Generic Multi-Compartmental CNS Distribution Model Structure for 9 Drugs Allows Prediction of Human Brain Target Site Concentrations

    NARCIS (Netherlands)

    Yamamoto, Yumi; Valitalo, Pyry A.; van den Berg, Dirk-Jan; Hartman, Robin; van den Brink, Willem; Wong, Yin Cheong; Huntjens, Dymphy R.; Proost, Johannes H.; Vermeulen, An; Krauwinkel, Walter; Bakshi, Suruchi; Aranzana-Climent, Vincent; Marchand, Sandrine; Dahyot-Fizelier, Claire; Couet, William; Danhof, Meindert; van Hasselt, Johan G. C.; de lange, Elizabeth C. M.

    Purpose Predicting target site drug concentration in the brain is of key importance for the successful development of drugs acting on the central nervous system. We propose a generic mathematical model to describe the pharmacokinetics in brain compartments, and apply this model to predict human

  8. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE......-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR...

  9. Three new human skulls from the Sima de los Huesos Middle Pleistocene site in Sierra de Atapuerca, Spain.

    Science.gov (United States)

    Arsuaga, J L; Martínez, I; Gracia, A; Carretero, J M; Carbonell, E

    1993-04-08

    Three important fossil hominids were found in July 1992 in the Middle Pleistocene cave site called Sima de los Huesos (Sierra de Atapuerca, Burgos, Northern Spain). One is a complete calvaria (cranium 4), the second a virtually complete cranium (cranium 5), the third represents a more fragmentary cranium of an immature individual (cranium 6). There is a large difference in size between the two adult specimens (for example endocranial volume 1,125 cm3 versus 1,390 cm3). The Atapuerca human remains are dated to > 300,000 years. The Atapuerca cranial sample fits within the 'archaic Homo sapiens' group, but is well differentiated from the Asian Homo erectus group. The extensive Atapuerca human collection is the most complete sample of Middle Pleistocene humans yet discovered from one site, and appears to document an early stage in Neanderthal evolution.

  10. The genome of a Late Pleistocene human from a Clovis burial site in western Montana

    KAUST Repository

    Rasmussen, Morten Arendt Rendt

    2014-02-12

    Clovis, with its distinctive biface, blade and osseous technologies, is the oldest widespread archaeological complex defined in North America, dating from 11,100 to 10,700 14 C years before present (bp) (13,000 to 12,600 calendar years bp). Nearly 50 years of archaeological research point to the Clovis complex as having developed south of the North American ice sheets from an ancestral technology. However, both the origins and the genetic legacy of the people who manufactured Clovis tools remain under debate. It is generally believed that these people ultimately derived from Asia and were directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western Montana. The human bones date to 10,705 ± 35 14 C years bp (approximately 12,707-12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal\\'ta population into Native American ancestors is also shared by the Anzick-1 individual and thus happened before 12,600 years bp. We also show that the Anzick-1 individual is more closely related to all indigenous American populations than to any other group. Our data are compatible with the hypothesis that Anzick-1 belonged to a population directly ancestral to many contemporary Native Americans. Finally, we find evidence of a deep divergence in Native American populations that predates the Anzick-1 individual. © 2014 Macmillan Publishers Limited. All rights reserved.

  11. Plutonium and uranium in human bones from areas surrounding the Semipalatinsk nuclear test site.

    Science.gov (United States)

    Yamamoto, Masayoshi; Hoshi, Masaharu; Sakaguchi, Aya; Shinohara, Kunihiko; Kurihara, Osamu; Apsalikov, Kazbek N; Gusev, Boris I

    2006-02-01

    To evaluate the present levels of 239,240Pu and U in residents living near the Semipalatinsk nuclear test site, more than 70 bone samples were obtained at autopsy. The subjects ranged in age from 30 to 86 years (mean 59.3+/-12.9). Most of the samples consisted of victims who died of various diseases. Plutonium and U were radiochemically separated and determined by alpha-ray spectrometry. The mean concentrations of 239,240Pu and 238U observed were 0.050+/-0.041 mBq/g-ash (vertebrae 71, long-bones 18) and 0.28+/-0.13 mBq/g-ash (22.8+/-10.6 microg U/kg-ash) (vertebrae 58, long bones 16), respectively. The present 239,240Pu levels were within the range found for human bone samples from other countries due solely to global fallout in the early 1980s. The average U concentration was close to the estimate (mean 22.5 microg U/kg-ash) for the UK, and about 10 times higher than those estimated for residents in New York City and Japan. By assuming that the average concentration of 239,240Pu in bone samples is the value at 45 years after instantaneous inhalation in 1955, the initial total intake and the effective dose for 45 years were estimated as 10 Bq and 0.2 mSv, respectively. The annual intake of total U (234,235,238U) and its effective dose for 60 years were estimated as 30 Bq for adult and 0.1 mSv, respectively, for chronic ingestion.

  12. Analysis of growth hormone and lactogenic binding sites cross-linked to iodinated human growth hormone

    International Nuclear Information System (INIS)

    Hughes, J.P.; Simpson, J.S.; Friesen, H.G.

    1983-01-01

    GH (GHR) and lactogenic receptors were analyzed after use of the cross-linking reagent ethylene glycol bis-(succinimidyl succinate) to attach covalently iodinated human GH (hGH) to binding proteins 1) on intact IM-9 lymphocytes, 2) in a partially purified GHR preparation from rabbit liver, and 3) in crude microsomal fractions from rabbit liver, rabbit mammary gland, and rat liver. The latter two microsomal preparations contain primarily lactogenic receptors, whereas in IM-9 lymphocytes and the rabbit liver preparations, GHR predominate. Cross-linked [125I]hGH-receptor complexes were solubilized, reduced, and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of proteins cross-linked to [125I]hGH in the microsomal fraction from rabbit liver showed a specifically labeled complex with an estimated molecular weight (mol wt) of 75K. A slightly lower mol wt (71K) was determined for the complex labeled in the purified GHR preparation. In contrast to the relatively low mol wt complexes in rabbit liver, a complex that migrated with an apparent mol wt of 130K was identified in IM-9 lymphocytes. Labeled complexes were identified at 66K from rat liver and 61K from rabbit mammary gland. If it is assumed that hGH contributes 21K to the mol wt of the radiolabeled complexes, then the approximate mol wts of hGH-binding sites are 50-54K from rabbit liver, 109K from IM-9 lymphocytes, 45K from rat liver, and 40K from rabbit mammary gland

  13. Quantification of Mesenchymal Stem Cells (MSCs) at sites of human prostate cancer.

    Science.gov (United States)

    Brennen, W Nathaniel; Chen, Shuangling; Denmeade, Samuel R; Isaacs, John T

    2013-01-01

    Circulating bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have an innate tropism for tumor tissue in response to the inflammatory microenvironment present in malignant lesions. The prostate is bombarded by numerous infectious and inflammatory insults over a lifetime. Chronic inflammation is associated with CXCL12, CCL5, and CCL2, which are highly overexpressed in prostate cancer. Among other cell types, these chemoattractant stimuli recruit BM-MSCs to the tumor. MSCs are minimally defined as plastic-adhering cells characterized by the expression of CD90, CD73, and CD105 in the absence of hematopoietic markers, which can differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs are immunoprivileged and have been implicated in tumorigenesis through multiple mechanisms, including promoting proliferation, angiogenesis, and metastasis, in addition to the generation of an immunosuppressive microenvironment. We have demonstrated that MSCs represent 0.01-1.1% of the total cells present in core biopsies from primary human prostatectomies. Importantly, these analyses were performed on samples prior to expansion in tissue culture. MSCs in these prostatectomy samples are FAP-, CD90-, CD73-, and CD105-positive, and CD14-, CD20-, CD34-, CD45-, and HLA-DR-negative. Additionally, like BM-MSCs, these prostate cancer-derived stromal cells (PrCSCs) were shown to differentiate into osteoblasts, adipocytes and chondrocytes. In contrast to primary prostate cancer-derived epithelial cells, fluorescently-labeled PrCSCs and BM-MSCs were both shown to home to CWR22RH prostate cancer xenografts following IV injection. These studies demonstrate that not only are MSCs present in sites of prostate cancer where they may contribute to carcinogenesis, but these cells may also potentially be used to deliver cytotoxic or imaging agents for therapeutic and/or diagnostic purposes.

  14. Distinct parietal sites mediate the influences of mood, arousal, and their interaction on human recognition memory.

    Science.gov (United States)

    Greene, Ciara M; Flannery, Oliver; Soto, David

    2014-12-01

    The two dimensions of emotion, mood valence and arousal, have independent effects on recognition memory. At present, however, it is not clear how those effects are reflected in the human brain. Previous research in this area has generally dealt with memory for emotionally valenced or arousing stimuli, but the manner in which interacting mood and arousal states modulate responses in memory substrates remains poorly understood. We investigated memory for emotionally neutral items while independently manipulating mood valence and arousal state by means of music exposure. Four emotional conditions were created: positive mood/high arousal, positive mood/low arousal, negative mood/high arousal, and negative mood/low arousal. We observed distinct effects of mood valence and arousal in parietal substrates of recognition memory. Positive mood increased activity in ventral posterior parietal cortex (PPC) and orbitofrontal cortex, whereas arousal condition modulated activity in dorsal PPC and the posterior cingulate. An interaction between valence and arousal was observed in left ventral PPC, notably in a parietal area distinct from the those identified for the main effects, with a stronger effect of mood on recognition memory responses here under conditions of relative high versus low arousal. We interpreted the PPC activations in terms of the attention-to-memory hypothesis: Increased arousal may lead to increased top-down control of memory, and hence dorsal PPC activation, whereas positive mood valence may result in increased activity in ventral PPC regions associated with bottom-up attention to memory. These findings indicate that distinct parietal sites mediate the influences of mood, arousal, and their interplay during recognition memory.

  15. The genome of a Late Pleistocene human from a Clovis burial site in western Montana

    KAUST Repository

    Rasmussen, Morten Arendt Rendt; Anzick, Sarah L.; Waters, Michael R.; Skoglund, Pontus; Degiorgio, Michael; Stafford, Thomas W Jr; Rasmussen, Simon B.; Moltke, Ida; Albrechtsen, Anders; Doyle, Shane M.; Poznik, G. David; Gudmundsdottir, Valborg; Yadav, Rachita; Malaspinas, Anna Sapfo; Samuel Stockton White, V.; Allentoft, Morten Erik; Cornejo, Omar Eduardo; Tambets, Kristiina; Eriksson, Anders; Heintzman, Peter D.; Karmin, Monika; Korneliussen, Thorfinn Sand; Meltzer, David J.; Pierre, Tracey L.; Stenderup, Jesper T.; Saag, Lauri; Warmuth, Vera M.; Lopes, Margarida C.; Malhi, Ripan Singh; Brunak, Sö ren; Sicheritz-Ponté n, Thomas; Barnes, Ian; Collins, Matthew J.; Orlando, Ludovic A A; Balloux, Franç ois; Manica, Andrea; Gupta, Ramneek; Metspalu, Mait; Bustamante, Carlos D.; Jakobsson, Mattias; Nielsen, Rasmus Wedel; Willerslev, Eske

    2014-01-01

    Clovis, with its distinctive biface, blade and osseous technologies, is the oldest widespread archaeological complex defined in North America, dating from 11,100 to 10,700 14 C years before present (bp) (13,000 to 12,600 calendar years bp). Nearly 50 years of archaeological research point to the Clovis complex as having developed south of the North American ice sheets from an ancestral technology. However, both the origins and the genetic legacy of the people who manufactured Clovis tools remain under debate. It is generally believed that these people ultimately derived from Asia and were directly related to contemporary Native Americans. An alternative, Solutrean, hypothesis posits that the Clovis predecessors emigrated from southwestern Europe during the Last Glacial Maximum. Here we report the genome sequence of a male infant (Anzick-1) recovered from the Anzick burial site in western Montana. The human bones date to 10,705 ± 35 14 C years bp (approximately 12,707-12,556 calendar years bp) and were directly associated with Clovis tools. We sequenced the genome to an average depth of 14.4× and show that the gene flow from the Siberian Upper Palaeolithic Mal'ta population into Native American ancestors is also shared by the Anzick-1 individual and thus happened before 12,600 years bp. We also show that the Anzick-1 individual is more closely related to all indigenous American populations than to any other group. Our data are compatible with the hypothesis that Anzick-1 belonged to a population directly ancestral to many contemporary Native Americans. Finally, we find evidence of a deep divergence in Native American populations that predates the Anzick-1 individual. © 2014 Macmillan Publishers Limited. All rights reserved.

  16. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    Science.gov (United States)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  17. Alterations in polyadenylation and its implications for endocrine disease

    DEFF Research Database (Denmark)

    Rehfeld, Anders Aagaard; Plass, Mireya; Krogh, Anders

    2013-01-01

    Introduction: Polyadenylation is the process in which the pre-mRNA is cleaved at the poly(A) site and a poly(A) tail is added - a process necessary for normal mRNA formation. Genes with multiple poly(A) sites can undergo alternative polyadenylation (APA), producing distinct mRNA isoforms with dif......Introduction: Polyadenylation is the process in which the pre-mRNA is cleaved at the poly(A) site and a poly(A) tail is added - a process necessary for normal mRNA formation. Genes with multiple poly(A) sites can undergo alternative polyadenylation (APA), producing distinct mRNA isoforms...... with different 3' untranslated regions (3' UTRs) and in some cases different coding regions. Two thirds of all human genes undergo APA. The efficiency of the polyadenylation process regulates gene expression and APA plays an important part in post-transcriptional regulation, as the 3' UTR contains various cis...

  18. Summary of Natural Resources that Potentially Influence Human Intrusion at the Area 5 Radioactive Waste Management Site, Nevada Test Site, Nye County, Nevada

    International Nuclear Information System (INIS)

    NSTec Environmental Management

    2007-01-01

    In 1993, Raytheon Services Nevada completed a review of natural resource literature and other sources to identify potentially exploitable resources and potential future land uses near the Area 5 Radioactive Waste Management Site (RWMS) of the Nevada Test Site (NTS), Nye County, Nevada, that could lead to future inadvertent human intrusion and subsequent release of radionuclides to the accessible environment. National Security Technologies, LLC, revised the original limited-distribution document to conform to current editorial standards and U.S. Department of Energy requirements for public release. The researchers examined the potential for future development of sand, gravel, mineral, petroleum, water resources, and rural land uses, such as agriculture, grazing, and hunting. The study was part of the performance assessment for Greater Confinement Disposal boreholes. Sand and gravel are not considered exploitable site resources because the materials are common throughout the area and the quality at the Area 5 RWMS is not ideal for typical commercial uses. Site information also indicates a very low mineral potential for the area. None of the 23 mining districts in southern Nye County report occurrences of economic mineral deposits in unconsolidated alluvium. The potential for oil and natural gas is low for southern Nye County. No occurrences of coal, tar sand, or oil shale on the NTS are reported in available literature. Several potential future uses of water were considered. Agricultural irrigation is impractical due to poor soils and existing water supply regulations. Use of water for geothermal energy development is unlikely because temperatures are too low for typical commercial applications using current technology. Human consumption of water has the most potential for cause of intrusion. The economics of future water needs may create a demand for the development of deep carbonate aquifers in the region. However, the Area 5 RWMS is not an optimal location for

  19. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Science.gov (United States)

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-08

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

  20. Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of the Human Origin Recognition Complex

    National Research Council Canada - National Science Library

    Mendez, Juan

    2000-01-01

    ... of cellular life tipically lost in cancer. In order to unravel the molecular mechanisms of human DNA replication in normal and cancer cells, we have started a search for human DNA sequences that serve as replicators", this is, binding sites...

  1. ANTHROPOGENIC POLLEN INDICATORS (API FROM ARCHAEOLOGICAL SITES AS LOCAL EVIDENCE OF HUMAN-INDUCED ENVIRONMENTS IN THE ITALIAN PENINSULA

    Directory of Open Access Journals (Sweden)

    A. M. Mercuri

    2013-04-01

    Full Text Available Pollen data from twenty-six archaeological sites are reviewed to investigate the development of human-induced environments through the presence of selected Anthropogenic Pollen Indicators (API. The sites are located in six Italian regions - Veneto, Emilia Romagna, Tuscany, Basilicata, Calabria, and Sicily - and in the Republic of San Marino. Their chronology spans from the Bronze to the Renaissance ages, from approximately 4200 to 500 years BP. The API which are common in these sites are properly considered important markers of human activity and anthropization in the Mediterranean area. The most frequent API taxa in pollen spectra are seven: Artemisia, Centaurea, Cichorieae and Plantago are ubiquitous and therefore they have the major relevance, followed by cereals and Urtica, and by Trifolium type. The spread of plants producing these pollen grains is sometimes marked by high percentage values in pollen spectra. Pollen records show that, as expected, cereals and wild synanthropic herbs were widespread near archaeological sites but local differences are evident. Ecological and chrono-cultural reasons may be at the base of the observed differences. In general, the synanthropic plants well represent the xeric environments that developed as a result of the continuous human pressure and changes in soil compositions. These changes have occurred especially during the mid and late Holocene.

  2. Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

    Science.gov (United States)

    Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

    2011-01-01

    In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

  3. Modelling multi-site transmission of the human papillomavirus and its impact on vaccination effectiveness

    Directory of Open Access Journals (Sweden)

    P. Lemieux-Mellouki

    2017-12-01

    Conclusions: Modelling genital-site only transmission may overestimate vaccination impact if extragenital infections contribute to systemic natural immunity or underestimate vaccination impact if a high proportion of genital infections originate from extragenital infections. Under current understanding of heterosexual HPV transmission and immunity, a substantial bias from using uni-site models in predicting vaccination effectiveness against genital HPV infection is unlikely to occur.

  4. Background studies: human-induced effects on the evolution of shallow land burial sites for radioactive waste disposal

    International Nuclear Information System (INIS)

    1987-11-01

    This report presents the results of a programme of background research on the human-induced effects on the long term evolution of shallow disposal sites for low level radioactive wastes. The work is intended to support development and use of the TIME2 simulation code. Within the context of climatic change up to the next glacial maximum three areas are addressed: planning and legislative control over site usage, biosphere state changes and intrusion. An appendix presents a discussion of some planning aspects of radioactive waste disposal. (author)

  5. NcoI dimorphic site located 8kb 3' to the human apolipoprotein AIV (APOA4) gene

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, R T; Malloy, M J; Kane, J P; Frossard, P M

    1988-02-11

    pA4C3 a 0.5kb fragment from the 3' end of the human apolipoprotein AIV cDNA was isolated from a human intestine cDNA library and cloned into the EcoRI site of the plasmid pUC18. NcoI (CCATGG) (New England Biolabs) detects a single two-allele polymorphism with a band at either 18.6kb or at 12.6kb. The human apolipoprotein AI-CIII-AIV gene complex has been assigned to the long arm of chromosome 11 by Southern blot analysis of human-Chinese hamster cell hybrids. Co-dominant segregation was demonstrated in one family of six individuals.

  6. BanII dimorphic site located in the third intron of the human apolipoprotein AI (APOA1) gene

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, R T; Kresnak, M T; Frossard, P M

    1988-02-11

    A 0.7kb fragment generated by AvaII digestion of pBL13AI, a 0.965kb full-length human apolipoprotein AI cDNA was cloned into the EcoRI site of pBR322. The apoAI cDNA was isolated from a lambdagt10 human fetal liver cDNA library. BanII (GPuGCPyC) (International Biotechnologies, Inc.) identifies two invariant bands at 1122bp and 417bp, and a single two-allele polymorphism with bands at either 274bp or 452bp. The human apolipoprotein AI-CIII-AIV gene complex has been localized on the long arm of chromosome 11 by Southern blot analysis of human-chinese hamster cell hybrids. Co-dominant segregation has been observed in two families (13 individuals). The BanII restriction map was constructed from DNA sequence data of the human apoAI gene. The 452bp fragment is generated by the loss of a BanII dimorphic site in the third intron of the apoAI gene, between the 178bp and the 274bp fragments.

  7. Environmental Legionella spp. collected in urban test sites of South East Queensland, Australia, are virulent to human macrophages in vitro.

    Science.gov (United States)

    Lawrence, Amba; Eglezos, Sofroni; Huston, Wilhelmina

    2016-01-01

    Legionellae are frequent contaminants of potable water supplies, resulting in sporadic infections and occasional outbreaks. Isolates of Legionella were collected from urban test sites within South East Queensland and evaluated for their virulence potential in vitro. Two strains (from the species Legionella londiniensis and Legionella quinlivanii) were demonstrated to have the ability to infect human macrophages, while a strain from the species Legionella anisa did not maintain an infection over the same time course. This suggests that the spectrum of urban environmentally associated Legionella with potential to cause human disease might be greater than currently considered. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  8. Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

    International Nuclear Information System (INIS)

    Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

    1990-01-01

    Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

  9. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

    International Nuclear Information System (INIS)

    Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

    1987-01-01

    To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

  10. Pancreatic hormones are expressed on the surfaces of human and rat islet cells through exocytotic sites

    DEFF Research Database (Denmark)

    Larsson, L I; Hutton, J C; Madsen, O D

    1989-01-01

    . Electron microscopy reveals the labeling to occur at sites of exocytotic granule release, involving the surfaces of extruded granule cores. The surfaces of islet cells were labeled both by polyclonal and monoclonal antibodies, excluding that receptor-interacting, anti-idiotypic hormone antibodies were...... for these results. It is concluded that the staining reflects interactions between the appropriate antibodies and exocytotic sites of hormone release....

  11. Early Pleistocene archaeological occurrences at the Feiliang site, and the archaeology of human origins in the Nihewan Basin, North China.

    Directory of Open Access Journals (Sweden)

    Shuwen Pei

    Full Text Available The Early Pleistocene archaeological evidence from the fluvio-lacustrine sequence of the Nihewan Basin (North China offers an excellent opportunity to explore early human evolution and behavior in a temperate setting in East Asia, following the earliest 'Out of Africa'. Here we present the first comprehensive study of the Feiliang (FL site, with emphasis on the archaeological sequence, site integrity, and stone artifact assemblages. Magnetostratigraphic dating results show that early humans occupied the site ca. 1.2 Ma. Archaeological deposits were buried rapidly in primary context within shallow lake margin deposits, with only minor post-depositional disturbance from relatively low energy hydraulic forces. The FL lithic assemblage is characterized by a core and flake, Oldowan-like or Mode 1 technology, with a low degree of standardization, expedient knapping techniques, and casually retouched flakes. The bone assemblage suggests that hominin occupation of the FL site was in an open habitat of temperate grassland with areas of steppe and water. The main features of the FL assemblage are discussed in the context of the early Pleistocene archaeology of Nihewan, for which an assessment of current and future research is also presented.

  12. Monitoring human factor risk characteristics at nuclear legacy sites in northwest Russia in support of radiation safety regulation.

    Science.gov (United States)

    Scheblanov, V Y; Sneve, M K; Bobrov, A F

    2012-12-01

    This paper describes research aimed at improving regulatory supervision of radiation safety during work associated with the management of spent nuclear fuel and radioactive waste at legacy sites in northwest Russia through timely identification of employees presenting unfavourable human factor risk characteristics. The legacy sites of interest include sites of temporary storage now operated by SevRAO on behalf of Rosatom. The sites were previously operational bases for servicing nuclear powered submarines and are now subject to major remediation activities. These activities include hazardous operations for recovery of spent nuclear fuel and radioactive waste from sub-optimal storage conditions. The paper describes the results of analysis of methods, procedures, techniques and informational issues leading to the development of an expert-diagnostic information system for monitoring of workers involved in carrying out the most hazardous operations. The system serves as a tool for human factor and professional reliability risk monitoring and has been tested in practical working environments and implemented as part of regulatory supervision. The work has been carried out by the Burnasyan Federal Medical Biophysical Center, within the framework of the regulatory cooperation programme between the Federal Medical-Biological Agency of Russia and the Norwegian Radiation Protection Authority.

  13. A ligand peptide motif selected from a cancer patient is a receptor-interacting site within human interleukin-11.

    Directory of Open Access Journals (Sweden)

    Marina Cardó-Vila

    Full Text Available Interleukin-11 (IL-11 is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC. Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i the peptide mimics a receptor-binding site within IL-11, (ii the binding of CGRRAGGSC to the IL-11R alpha is functionally relevant, (iii Arg4 and Ser8 are the key residues mediating the interaction, and (iv the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11R alpha has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs.

  14. Sites of instability in the human TCF3 (E2A) gene adopt G-quadruplex DNA structures in vitro

    Science.gov (United States)

    Williams, Jonathan D.; Fleetwood, Sara; Berroyer, Alexandra; Kim, Nayun; Larson, Erik D.

    2015-01-01

    The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination. PMID:26029241

  15. Monitoring human factor risk characteristics at nuclear legacy sites in northwest Russia in support of radiation safety regulation

    International Nuclear Information System (INIS)

    Scheblanov, V Y; Bobrov, A F; Sneve, M K

    2012-01-01

    This paper describes research aimed at improving regulatory supervision of radiation safety during work associated with the management of spent nuclear fuel and radioactive waste at legacy sites in northwest Russia through timely identification of employees presenting unfavourable human factor risk characteristics. The legacy sites of interest include sites of temporary storage now operated by SevRAO on behalf of Rosatom. The sites were previously operational bases for servicing nuclear powered submarines and are now subject to major remediation activities. These activities include hazardous operations for recovery of spent nuclear fuel and radioactive waste from sub-optimal storage conditions. The paper describes the results of analysis of methods, procedures, techniques and informational issues leading to the development of an expert-diagnostic information system for monitoring of workers involved in carrying out the most hazardous operations. The system serves as a tool for human factor and professional reliability risk monitoring and has been tested in practical working environments and implemented as part of regulatory supervision. The work has been carried out by the Burnasyan Federal Medical Biophysical Center, within the framework of the regulatory cooperation programme between the Federal Medical–Biological Agency of Russia and the Norwegian Radiation Protection Authority. (paper)

  16. Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

    International Nuclear Information System (INIS)

    Alderson, R.; Pastan, I.; Cheng, S.

    1985-01-01

    The binding of [ 125 I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[ 125 I]T3 (BrAc[ 125 I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (K /sub d/ = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (K /sub k/ = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [ 125 I]T3. The affinity labeling ligand BrAc[ 125 I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[ 125 I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000

  17. Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

    Science.gov (United States)

    Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

    2016-12-01

    Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Human tissue factor: cDNA sequence and chromosome localization of the gene

    International Nuclear Information System (INIS)

    Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

    1987-01-01

    A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

  19. Genome-wide profiling of H3K56 acetylation and transcription factor binding sites in human adipocytes.

    Directory of Open Access Journals (Sweden)

    Kinyui Alice Lo

    Full Text Available The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte

  20. Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

    Science.gov (United States)

    Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

    2016-07-01

    Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

  1. Palaeoloxodon and Human Interaction: Depositional Setting, Chronology and Archaeology at the Middle Pleistocene Ficoncella Site (Tarquinia, Italy)

    Science.gov (United States)

    Aureli, Daniele; Contardi, Antonio; Giaccio, Biagio; Jicha, Brian; Lemorini, Cristina; Madonna, Sergio; Magri, Donatella; Marano, Federica; Milli, Salvatore; Modesti, Valerio; Palombo, Maria Rita; Rocca, Roxane

    2015-01-01

    The Ficoncella site in northern Latium (Italy) represents a unique opportunity to investigate the modalities of a short occupation in an alluvial setting during the Lower Palaeolithic. The small excavation area yielded a lithic assemblage, a carcass of Palaeoloxodon antiquus, and some other faunal remains. The main objectives of the study are to better characterize the depositional context where the Palaeoloxodon and the lithic assemblage occur, and to evaluate with greater precision the occupation dynamics. A 25 m-long well was drilled just above the top of the terrace of the Ficoncella site and faunal and lithic remains were analyzed with current and innovative techniques. The archaeological site contains floodplain deposits as it is located next to a small incised valley that feeds into a larger valley of the Mignone River. A tephra layer capping the site is 40Ar/39Ar dated to 441± 8 ka. Collectively, the geochronologic, tephrochronologic and geologic data, suggest the site was occupied during MIS 13. The new results should prompt further research at Ficoncella in order to improve our understanding of the dynamics of human settlement in Europe during the Early to Middle Pleistocene. PMID:25898322

  2. Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

    Science.gov (United States)

    Abou-Zied, Osama K

    2015-01-01

    Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.

  3. In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: Implications for biological function

    DEFF Research Database (Denmark)

    Ahmad, I.; Hoessli, D.C.; Gupta, Ramneek

    2007-01-01

    Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence...... both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes...... of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should...

  4. Human hyoid bones from the middle Pleistocene site of the Sima de los Huesos (Sierra de Atapuerca, Spain).

    Science.gov (United States)

    Martínez, I; Arsuaga, J L; Quam, R; Carretero, J M; Gracia, A; Rodríguez, L

    2008-01-01

    This study describes and compares two hyoid bones from the middle Pleistocene site of the Sima de los Huesos in the Sierra de Atapuerca (Spain). The Atapuerca SH hyoids are humanlike in both their morphology and dimensions, and they clearly differ from the hyoid bones of chimpanzees and Australopithecus afarensis. Their comparison with the Neandertal specimens Kebara 2 and SDR-034 makes it possible to begin to approach the question of temporal variation and sexual dimorphism in this bone in fossil humans. The results presented here show that the degree of metric and anatomical variation in the fossil sample was similar in magnitude and kind to living humans. Modern hyoid morphology was present by at least 530 kya and appears to represent a shared derived feature of the modern human and Neandertal evolutionary lineages inherited from their last common ancestor.

  5. The human nuclear poly(a-binding protein promotes RNA hyperadenylation and decay.

    Directory of Open Access Journals (Sweden)

    Stefan M Bresson

    Full Text Available Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A-binding protein (PABPN1, the poly(A polymerases (PAPs, PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

  6. Role of strategic human resource management in crisis management in Australian greenfield hospital sites: a crisis management theory perspective.

    Science.gov (United States)

    Kendrick, Madeleine Iris; Bartram, Timothy; Cavanagh, Jillian; Burgess, John

    2017-11-20

    Objective This study examined strategic human resource management (SHRM) activities in two case hospitals relative to their approach to greenfield site success. Methods A comparative case study analysis approach was used, with documents sourced from public, open-access sites. The theoretical framework of crisis management theory's (CMT) proactive management and open communication channels was used to examine the documents, which were annual reports addressing both hospitals' first year of performance, union publications and transcripts of relevant parliamentary inquiries. Results The hospital that effectively used CMT in its first 12 months was demonstratively more 'successful' than the hospital that reported to not have effectively used CMT. 'Success' in this project was articulated as the hospital's ability to consolidate operations, without ongoing negative media attention, after 12 months. Conclusion This study provided an identification of how the use of CMT in a hospital's greenfield stage can increase the hospital's chances of 'success'. What is known about the topic? Journal and media articles illustrated a gap in greenfield human resource management (HRM) regarding successful consolidation, especially the healthcare context. Although manufacturing firms are addressed in academic literature in a greenfield context, there is a lack of knowledge concerning successful greenfield HRM in a healthcare context. What does this paper add? This study is among the first to identify the role of CMT in successful greenfield site establishment by identifying its presence in management activities. What are the implications for practitioners? The findings of this study suggest a potential link between the implementation of CMT and greenfield site success. This could allow future greenfield healthcare sites to operate with less cost and risk. The lack of stakeholder participation in the present study limits the applicability of its findings. However, archival document

  7. Discovery and development of inhibitors selective for human constitutive proteasome and immunoproteasome active sites

    NARCIS (Netherlands)

    Xin, B.

    2017-01-01

    This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally

  8. Ensemble approach combining multiple methods improves human transcription start site prediction.

    LENUS (Irish Health Repository)

    Dineen, David G

    2010-01-01

    The computational prediction of transcription start sites is an important unsolved problem. Some recent progress has been made, but many promoters, particularly those not associated with CpG islands, are still difficult to locate using current methods. These methods use different features and training sets, along with a variety of machine learning techniques and result in different prediction sets.

  9. Human Detection and Classification of Landing Sites for Search and Rescue Drones

    NARCIS (Netherlands)

    N. Martins, Felipe; de Groot, Marc; Stokkel, Xeryus; Wiering, Marco

    2016-01-01

    Search and rescue is often time and labour intensive. We present a system to be used in drones to make search and rescue operations more effective. The system uses a drone downward facing camera to detect people and to evaluate potential sites as being safe or not for the drone to land. Histogram of

  10. Insulin stimulation regulates AS160 and TBC1D1 phosphorylation sites in human skeletal muscle

    DEFF Research Database (Denmark)

    Middelbeek, R J W; Chambers, M A; Tantiwong, P

    2013-01-01

    Individuals with obesity and type 2 diabetes (T2D) are typically insulin resistant, exhibiting impaired skeletal muscle glucose uptake. Animal and cell culture experiments have shown that site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 and TBC1D1 is critical for GLUT4 tr...

  11. Conformational dynamics of bacterial and human cytoplasmic models of the ribosomal A-site

    Czech Academy of Sciences Publication Activity Database

    Panecka, J.; Šponer, Jiří; Trylska, J.

    112C, MAY 2015 (2015), s. 96-110 ISSN 0300-9084 R&D Projects: GA ČR(CZ) GBP305/12/G034; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS * DECODING SITE * CRYSTAL-STRUCTURE Subject RIV: BO - Biophysics Impact factor: 3.017, year: 2015

  12. Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

    Directory of Open Access Journals (Sweden)

    Turgut Bastug

    Full Text Available Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+ ions, one H(+ and the counter-transport of one K(+ ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph, whose three dimensional structure is known is also coupled to three Na(+ ions but only two Na(+ ion binding sites have been observed in the crystal structure of Glt(Ph. In order to fully utilize the Glt(Ph structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph and accurately determine the number and location of Na(+ ions coupled to transport. Several sites have been proposed for the binding of a third Na(+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph and reveal a new site for the third Na(+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+ ion in Glt(Ph and EAAT1.

  13. Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7

    Science.gov (United States)

    Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W. H.; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2010-01-01

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, 163DVD165 and 221FWGWGREDDE230, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp224 as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp228 acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics. PMID:20843813

  14. Distinct pools of cdc25C are phosphorylated on specific TP sites and differentially localized in human mitotic cells.

    Directory of Open Access Journals (Sweden)

    Celine Franckhauser

    Full Text Available BACKGROUND: The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1. METHODOLOGY/PRINCIPAL FINDINGS: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis. CONCLUSIONS/SIGNIFICANCE: These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C

  15. Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation.

    Science.gov (United States)

    de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Merlo, Kleison C; Romão, Tatiany P; Rocha, Pollyanna O; Assis, Ludmila A; Nascimento, Larissa M; Xavier, Camila C; Rezende, Antonio M; Reis, Christian R S; Papadopoulou, Barbara

    2018-03-23

    The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.

  16. CKI isoforms α and ε regulate Star–PAP target messages by controlling Star–PAP poly(A) polymerase activity and phosphoinositide stimulation

    Science.gov (United States)

    Laishram, Rakesh S.; Barlow, Christy A.; Anderson, Richard A.

    2011-01-01

    Star–PAP is a non-canonical, nuclear poly(A) polymerase (PAP) that is regulated by the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PI4,5P2), and is required for the expression of a select set of mRNAs. It was previously reported that a PI4,5P2 sensitive CKI isoform, CKIα associates with and phosphorylates Star–PAP in its catalytic domain. Here, we show that the oxidative stress-induced by tBHQ treatment stimulates the CKI mediated phosphorylation of Star–PAP, which is critical for both its polyadenylation activity and stimulation by PI4,5P2. CKI activity was required for the expression and efficient 3′-end processing of its target mRNAs in vivo as well as the polyadenylation activity of Star–PAP in vitro. Specific CKI activity inhibitors (IC261 and CKI7) block in vivo Star–PAP activity, but the knockdown of CKIα did not equivalently inhibit the expression of Star–PAP targets. We show that in addition to CKIα, Star–PAP associates with another CKI isoform, CKIε in the Star–PAP complex that phosphorylates Star–PAP and complements the loss of CKIα. Knockdown of both CKI isoforms (α and ε) resulted in the loss of expression and the 3′-end processing of Star–PAP targets similar to the CKI activity inhibitors. Our results demonstrate that CKI isoforms α and ε modulate Star–PAP activity and regulates Star–PAP target messages. PMID:21729869

  17. Neuroleptics and β-carbolines displace (3H)imipramine from its binding sites in human and rat tissues

    International Nuclear Information System (INIS)

    Rommelspacher, H.; Strauss, S.

    1985-01-01

    Most investigations dealing with the pharmacological characterization of ( 3 H)imipramine binding sites focus on tricyclic antidepressants (TCA). This approach seemed to be justified since imipramine belongs to that chemical group. Langer and coworkers, however, introduced a tetrahydro-β-carboline (THβC) as a possible endogenous ligand. Thus, the high affinity of imipramine towards the binding sites might not be due to its special chemical structure but due to its tricyclic nature. In the present paper the structure-activity-relationships of neuroleptics and β-carbolines were investigated and compared with that of tricyclic antidepressants. Among the tricyclic neuroleptics those with an electron attracting substituent (-Cl) exerted highest affinity. The effect was attenuated by a long, cyclic side chain. The affinity of tricyclic neuroleptics was only slightly weaker than that of 6-Meo-THβC the suggested endogenous ligand. The experiments with other THβCs supported the observation that an electron attracting substituent increases the affinity of a compound to the ( 3 H)imipramine binding sites. Comparison of the binding characteristics of ( 3 H)imipramine to membranes of human brain and thrombocytes as well as those of rat brain and thrombocytes revealed no differences among both species. Furthermore, the displacing potencies of neuroleptics were very similar with only slightly more activity in human tissue. As a methodological aspect the applicability of the 'Lowry' method to determine the protein concentration is discussed. (Author)

  18. Modeling human exposure to hazardous-waste sites: a question of completeness

    International Nuclear Information System (INIS)

    Daniels, J.I.; McKone, T.E.

    1991-01-01

    In risk analysis, we use human-exposure assessments to translate contaminant sources into quantitative estimates of the amount of contaminant that comes in contact with human-environment boundaries, that is, the lungs, the gastrointestinal tract, and the skin surface of individuals within a specified population. An assessment of intake requires that we determine how much crosses these boundaries. Exposure assessments often rely implicitly in the assumption that exposure can be linked by simple parameters to ambient concentration in air, water, and soil. However, more realistic exposure models require that we abandon such simple assumptions. To link contaminant concentrations in water, air, or soil with potential human intakes, we constrict pathway-exposure factors (PEFs). For each PEF we combine information in environmental partitioning as well as human anatomy, physiology, and patterns into an algebraic term that converts concentrations of contaminants (in mg/L water, mg/m 3 air, and mg/kg soil) into a daily intake per unit body weight in mg/kg-d for a specific rout of exposure such as inhalation, ingestion, or dermal uptake. Using examples involving human exposure to either a radionuclide (tritium, 3 H) or a toxic organic chemical (tetrachloroethylene, PCE) in soil, water, and air, we illustrate the use of PEFs and consider the implications for risk assessment. (au)

  19. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  20. Natural selection affects multiple aspects of genetic variation at putatively peutral sites across the human genome

    DEFF Research Database (Denmark)

    Lohmueller, Kirk E; Albrechtsen, Anders; Li, Yingrui

    2011-01-01

    A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries...... these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination...... and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations...

  1. Lessons learned: Needs for improving human health risk assessment at USDOE Sites

    International Nuclear Information System (INIS)

    Hamilton, L.D.; Holtzman, S.; Meinhold, A.F.; Morris, S.C.; Rowe, M.D.; Daniels, J.I.; Layton, D.W.; Anspaugh, L.R.

    1993-09-01

    Realistic health risk assessments were performed in a pilot study of three U.S. Department of Energy (USDOE) sites. These assessments, covering a broad spectrum of data and methods, were used to identify needs for improving future health risk assessments at USDOE sites. Topics receiving specific recommendations for additional research include: choice of distributions for Monte Carlo simulation; estimation of risk reduction; analysis of the U.S. Department of Agriculture Database on food and nutrient intakes; investigations on effects of food processing on contaminant levels; background food and environmental concentrations of contaminants; method for handling exposures to groundwater plumes, methods for analyzing less than lifetime exposure to carcinogens; and improvement of bioaccumulation factors

  2. Geological activity of humans represented in the world heritage sites of India, Italy, and Russia: Evidence of the anthropocene

    Directory of Open Access Journals (Sweden)

    Ansari M K.

    2016-01-01

    Full Text Available The idea of the Anthropocene attracts attention of scientists, policy-makers, and broad public to the geological activity of humans and poses new important questions for the modern stratigraphy. The growth of the Anthropocene-related knowledge and its promotion can be based potentially on the UNESCO World Heritage Sites (WHS. On the one hand, many of these sites provide spectacular evidence of the human activity. On the other hand, these are remarkable tourist attractions. The WHSs of three heritage-rich countries, namely India, Italy, and Russia, have been assessed with regard to how these reflect the geological activity of humans. It is established that 65-90% of all WHSs in each country provide direct and indirect evidence of such an activity (artificial caves, terrace building, etc., which appears to be enough for the general discussion of the idea of the Anthropocene. However, the distribution of the WHSs by their age allows focusing only on the “early” (before 1800 AD start of the Anthropocene, which is not enough for full discussion of the lower limit of this unit. The examples considered in the present study imply that some WHSs alone provide very important pieces of the Anthropocene-related knowledge.

  3. Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site

    International Nuclear Information System (INIS)

    Collins, C.J.; Underdahl, J.P.; Levene, R.B.; Ravera, C.P.; Morin, M.J.; Dombalagian, M.J.; Ricca, G.; Livingston, D.M.; Lynch, D.C.

    1987-01-01

    A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for van Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span ≅ 175 kilobases of human DNA sequence, and hybridization analysis suggest that the vWf coding unit is ≅150 kilobases in length. Within one of these clones, the vWF transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWF translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species

  4. A functional MiR-124 binding-site polymorphism in IQGAP1 affects human cognitive performance.

    Directory of Open Access Journals (Sweden)

    Lixin Yang

    Full Text Available As a product of the unique evolution of the human brain, human cognitive performance is largely a collection of heritable traits. Rather surprisingly, to date there have been no reported cases to highlight genes that underwent adaptive evolution in humans and which carry polymorphisms that have a marked effect on cognitive performance. IQ motif containing GTPase activating protein 1 (IQGAP1, a scaffold protein, affects learning and memory in a dose-dependent manner. Its expression is regulated by miR-124 through the binding sites in the 3'UTR, where a SNP (rs1042538 exists in the core-binding motif. Here we showed that this SNP can influence the miR-target interaction both in vitro and in vivo. Individuals carrying the derived T alleles have higher IQGAP1 expression in the brain as compared to the ancestral A allele carriers. We observed a significant and male-specific association between rs1042538 and tactile performances in two independent cohorts. Males with the derived allele displayed higher tactual performances as compared to those with the ancestral allele. Furthermore, we found a highly diverged allele-frequency distribution of rs1042538 among world human populations, likely caused by natural selection and/or recent population expansion. These results suggest that current human populations still carry sequence variations that affect cognitive performances and that these genetic variants may likely have been subject to comparatively recent natural selection.

  5. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase.

    Science.gov (United States)

    Wang, Xianwei; Zhang, John Z H; He, Xiao

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  6. Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

    International Nuclear Information System (INIS)

    Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

    1986-01-01

    Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

  7. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xianwei [Center for Optics and Optoelectronics Research, College of Science, Zhejiang University of Technology, Hangzhou, Zhejiang 310023 (China); State Key Laboratory of Precision Spectroscopy, Institute of Theoretical and Computational Science, East China Normal University, Shanghai 200062 (China); Zhang, John Z. H.; He, Xiao, E-mail: xiaohe@phy.ecnu.edu.cn [State Key Laboratory of Precision Spectroscopy, Institute of Theoretical and Computational Science, East China Normal University, Shanghai 200062 (China); NYU-ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai 200062 (China)

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein’s internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  8. CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

    Science.gov (United States)

    Zhang, Zijun; Xing, Yi

    2017-09-19

    Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Location of the higher affinity copper site on human hemoglobin by the use of the spin label technique

    International Nuclear Information System (INIS)

    Tabak, M.; Louro, S.R.W.

    1983-11-01

    Addition of copper (II) ions to Cys β-93 maleimide spin-labelled human hemoglobin A produces a dramatic decrease in the amplitude of the spin-label ESR spectra. This effect was analyzed in the framework of Leigh's theory which permits interspin distances to be deduced from the effect of dipolar coupling on the ESR spectra and led to an estimate of 9A as the distance between the label and the higher affinity copper site. Taking into account the previous results which suggest that four nitrogen atoms coordinate with copper, and that the N terminal val β-1 and His β-2 residues are involved, the location of the higher affinity copper site is proposed to be at the β 1 β 2 interface of the hemoglobin molecule, involving the N terminal of one β subunit and the C terminal of the other. (Author) [pt

  10. Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

    1983-01-01

    Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

  11. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  12. Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

    NARCIS (Netherlands)

    Lai, Xuelei; Wichers, Harry J.; Soler-Lopez, Montserrat; Dijkstra, Bauke W.

    2017-01-01

    Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the

  13. Evidence for alpha-MSH binding sites on human scalp hair follicles: preliminary results

    NARCIS (Netherlands)

    Nanninga, P. B.; Ghanem, G. E.; Lejeune, F. J.; Bos, J. D.; Westerhof, W.

    1991-01-01

    Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond

  14. Prevalence of Human Papillomavirus in Anal and Oral Sites Among Patients with Genital Warts

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Sand, Carsten; Forslund, Ola

    2014-01-01

    Genital warts are caused by human papillomavirus (HPV). HPV is a leading cause of anogenital malignancies and a role of HPV in the aetiology of oro-pharyngeal cancers has been demonstrated. The frequency of oral HPV infection in patients with genital warts and the association between concomitant...

  15. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  16. A foetal tile from an archaeological site: anthropological investigation of human remains recovered in a medieval cemetery in Northern Italy.

    Science.gov (United States)

    Licata, Marta; Rossetti, Chiara; Tosi, Adelaide; Badino, Paola

    2018-06-01

    The recovery of foetal remains is very sporadic in archaeology, especially due the scarce degree of bone mineralisation. This paper presents the singular archaeological discovery of a foetal tile preserving the bone remains, object of our anthropological examination. The foetal tile was discovered during an archaeological excavation in a medieval site (Northern Italy). The tile was analysed by CT scan and later, human remains were anthropologically examined. The archaeological investigation revealed a special ritual destined to foetuses while forensic anthropological analysis allowed estimating the gestational age near to 21-24 weeks.

  17. Plutonium and Uranium in Human Bones from Areas surrounding the Semipalatinsk Nuclear Test Site

    OpenAIRE

    Masayoshi, YAMAMOTO; Masaharu, HOSHI; Aya, SAKAGUCHI; Kunihiko, SHINOHARA; Osamu, KURIHARA; Kazbek N., APSALIKOV; Boris I., GUSEV; Low Level Radioactivity Laboratory, K-INET, Kanazawa University; Research Institute for Radiation Biology and Medicine, Hiroshima University; Low Level Radioactivity Laboratory, K-INET, Kanazawa University; Japan Nuclear Cycle Development Institute; Japan Nuclear Cycle Development Institute; Kazakh Science Research Institute for Radiation, Medicine and Ecology; Kazakh Science Research Institute for Radiation, Medicine and Ecology

    2006-01-01

    To evaluate the present levels of^Pu and U in residents living near the Semipalatinsk nuclear test site, more than 70 bone samples were obtained at autopsy. The subjects ranged in age from 30 to 86 years (mean 59.3±12.9). Most of the samples consisted of victims who died of various diseases. Plutonium and U were radiochemically separated and determined by α-ray spectrometry. The mean concentrations of ^Pu and ^U observed were 0.050±0.041 mBq/g-ash (vertebrae 71, long-bones 18) and 0.28±0.13 m...

  18. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Lyon, David; Young, Clifford

    2017-01-01

    that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

  19. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

    International Nuclear Information System (INIS)

    Nobile, C.; Romeo, G.

    1988-01-01

    A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

  20. A genome-wide screen in human embryonic stem cells reveals novel sites of allele-specific histone modification associated with known disease loci

    LENUS (Irish Health Repository)

    Prendergast, James G D

    2012-05-19

    AbstractBackgroundChromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs).ResultsUsing a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders.ConclusionThese results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

  1. Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

    Energy Technology Data Exchange (ETDEWEB)

    Bijari, Nooshin [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Shokoohinia, Yalda [Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza; Ranjbar, Samira; Parvaneh, Shahram [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Moieni-Arya, Maryam [Student Research Committee, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2013-11-15

    The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp{sub 214} residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96 nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding. -- Highlights: • Hydrophobic interactions play an important role in osthole–HSA interaction. • Sudlow's I site is possible binding site of osthole. • Osthole inhibits esterase activity of HSA. • Osthole binding induces no gross protein structural changes.

  2. NGS-based approach to determine the presence of HPV and their sites of integration in human cancer genome.

    Science.gov (United States)

    Chandrani, P; Kulkarni, V; Iyer, P; Upadhyay, P; Chaubal, R; Das, P; Mulherkar, R; Singh, R; Dutt, A

    2015-06-09

    Human papilloma virus (HPV) accounts for the most common cause of all virus-associated human cancers. Here, we describe the first graphic user interface (GUI)-based automated tool 'HPVDetector', for non-computational biologists, exclusively for detection and annotation of the HPV genome based on next-generation sequencing data sets. We developed a custom-made reference genome that comprises of human chromosomes along with annotated genome of 143 HPV types as pseudochromosomes. The tool runs on a dual mode as defined by the user: a 'quick mode' to identify presence of HPV types and an 'integration mode' to determine genomic location for the site of integration. The input data can be a paired-end whole-exome, whole-genome or whole-transcriptome data set. The HPVDetector is available in public domain for download: http://www.actrec.gov.in/pi-webpages/AmitDutt/HPVdetector/HPVDetector.html. On the basis of our evaluation of 116 whole-exome, 23 whole-transcriptome and 2 whole-genome data, we were able to identify presence of HPV in 20 exomes and 4 transcriptomes of cervical and head and neck cancer tumour samples. Using the inbuilt annotation module of HPVDetector, we found predominant integration of viral gene E7, a known oncogene, at known 17q21, 3q27, 7q35, Xq28 and novel sites of integration in the human genome. Furthermore, co-infection with high-risk HPVs such as 16 and 31 were found to be mutually exclusive compared with low-risk HPV71. HPVDetector is a simple yet precise and robust tool for detecting HPV from tumour samples using variety of next-generation sequencing platforms including whole genome, whole exome and transcriptome. Two different modes (quick detection and integration mode) along with a GUI widen the usability of HPVDetector for biologists and clinicians with minimal computational knowledge.

  3. Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome.

    Science.gov (United States)

    Lohmueller, Kirk E; Albrechtsen, Anders; Li, Yingrui; Kim, Su Yeon; Korneliussen, Thorfinn; Vinckenbosch, Nicolas; Tian, Geng; Huerta-Sanchez, Emilia; Feder, Alison F; Grarup, Niels; Jørgensen, Torben; Jiang, Tao; Witte, Daniel R; Sandbæk, Annelli; Hellmann, Ines; Lauritzen, Torsten; Hansen, Torben; Pedersen, Oluf; Wang, Jun; Nielsen, Rasmus

    2011-10-01

    A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.

  4. Stress hormone levels in a freshwater turtle from sites differing in human activity.

    Science.gov (United States)

    Polich, Rebecca L

    2016-01-01

    Glucocorticoids, such as corticosterone (CORT), commonly serve as a measure of stress levels in vertebrate populations. These hormones have been implicated in regulation of feeding behaviour, locomotor activity, body mass, lipid metabolism and other crucial behaviours and physiological processes. Thus, understanding how glucocorticoids fluctuate seasonally and in response to specific stressors can yield insight into organismal health and the overall health of populations. I compared circulating CORT concentrations between two similar populations of painted turtle, Chrysemys picta, which differed primarily in the level of exposure to human recreational activities. I measured basal CORT concentrations as well as the CORT stress response and did not find any substantive difference between the two populations. This similarity may indicate that painted turtles are not stressed by the presence of humans during the nesting season. The results of this study contribute to our understanding of CORT concentrations in freshwater reptiles, a group that is historically under-represented in studies of circulating hormone concentrations; specifically, studies that seek to use circulating concentrations of stress hormones, such as CORT, as a measure of the effect of human activities on wild populations. They also give insight into how these species as a whole may respond to human recreational activities during crucial life-history stages, such as the nesting season. Although there was no discernable difference between circulating CORT concentrations between the urban and rural populations studied, I did find a significant difference in circulating CORT concentrations between male and female C. picta. This important finding provides better understanding of the sex differences between male and female painted turtles and adds to our understanding of this species and other species of freshwater turtle.

  5. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection Against Ebolaviruses

    Science.gov (United States)

    2017-03-31

    search, analyzed data, and wrote and/or edited the paper . E.G. and L.M.W. designed the germline-reverted constructs and E.G., L.M.W., A.Z.W., D.M.A... albumin (PBSA), and incubated with dilutions of test antibody (5, 50 nM). Bound Abs were detected with anti-human IgG conjugated to horseradish

  6. Two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Meager, A; Parti, S; Leung, H; Woolley, J; Peil, E; Sidhu, S; Roberts, T

    1987-11-23

    Three monoclonal antibodies (MoAbs L49-15, L81-11 and L238-14) were raised against recombinant human lymphotoxin (rLT) derived from E. coli containing the cDNA sequence specifying LT. These MoAbs were ideal reagents for immunoassay of LT and a very sensitive, highly specific immunoradiometric assay (IRMA) was developed. This assay was rapid to perform and was capable of detecting as little as 10 pg/ml of LT. Application of the LT IRMA in combination with previously developed human gamma-interferon (IFN-..gamma..) and human tumour necrosis factor (TNF)-specific IRMA permitted independent estimations of these three substances to be carried out in parallel. By these means, it was found that RPMI 1788 lymphoblastoid cell line produced both LT and TNF, but not IFN-..gamma... Extensive analyses on cytokine (monokine and lymphokine) preparations derived from a variety of activated lymphocytes are also reported. Co-production of LT, TNF, and IFN-..gamma.. was a common finding, even occurring in alloantigen-specific T helper cell clones. 45 refs.; 3 figs.; 4 tabs.

  7. Air quality assessment on human well-being in the vicinity of quarry site

    Science.gov (United States)

    Ibrahim, W. H. W.; Marinie, E.; Yunus, J.; Asra, N.; Sukor, K. Mohd

    2018-02-01

    This study aims to investigate the variation of air pollutants associated with the quarry activities prior to classified distance from quarry site. Air pollutants were monitored with the use of instruments which are Rae System Multirae Lite Pumped (PGM-6208) to measure indoor air quality while TSI 8533 Dusttrack Drx Desktop Aerosol Monitor to measure outdoor air quality. Sampling will be replicated two times. The locations of quarry are at Bandar Saujana Putra and Taman Kajang Perdana 2, Selangor. The objectives of this study are to investigate the impact of quarry mining by preparing the suitable Indoor Air Quality Index and to prepare preventive measure for residential that caused from quarry mining activities. Both Qualitative and Quantitative approaches will be implemented in this study, which employed case study and interview survey. Both quarries identified previously will be the main case study. The Respondent’s interviews are from Local Authority and Quarry Management Staff while questionnaire surveys from selected residences. Measurement method will be used to measure the Particle Matter (PM2.5) for indoor and outdoor in selected resident’s area. However, this paper is primed to discuss the method used in this study. It is not only presents the beneficial information for future research on methodologies employed but also it is anticipated the benefit to environment which can increased residents’ well-being in the vicinity of quarry sites.

  8. Platelet deposition at angioplasty sites and its relation to restenosis in human iliac and femoropopliteal arteries

    International Nuclear Information System (INIS)

    Minar, E.; Ehringer, H.; Ahmadi, R.; Dudczak, R.; Leitha, T.; Koppensteiner, R.; Jung, M.; Stuempflen, A.

    1989-01-01

    The amount and time course of platelet accumulation at angioplasty sites and influence of these platelets on restenosis after percutaneous transluminal angioplasty (PTA) in peripheral arteries were determined in 92 patients, who received either a high or low dose of aspirin. Platelet deposition was quantitated by means of dual-radiotracer scintigraphy and calculation of a platelet accumulation index (PAI). The PAI was higher (P less than .05) 4-6 hours after PTA compared with that on subsequent days. There was a trend toward greater platelet accumulation in vessels with extensive dissection. Platelet accumulation at the PTA site occurred with both doses of aspirin, with no differences between the two dosage groups. Twenty-one of 67 patients who underwent PTA in the femoropopliteal segment developed restenosis during a median follow-up of 14 months. The median PAI at 4-6 and 22-24 hours after PTA was significantly less in these 21 patients than in the 46 without restenosis. The data suggest that use of antiplatelet agents to prevent platelet deposition after PTA may not be useful for prevention of restenosis

  9. Na,K-ATPase binding sites in human erythrocytes in cirrhosis of the liver

    International Nuclear Information System (INIS)

    Schober, O.; Oetting, G.; Bossaller, C.

    1985-01-01

    The number of red blood cell ouabain binding sites, total-body potassium (TBK), serum potassium, exchangeable sodium, and serum sodium was studied in 24 patients with cirrhosis of the liver. The number of red cell ouabain binding sites, measured by equilibrium binding of 3 H-ouabain, showed a significant increase in the number of Na,K pumps in patients with cirrhosis of the liver (447+-99) as compared with a control group (281+-50, n=36). TBK was measured by counting the endogenous K-40 in a whole-body counter. TBK was 76+-10% in cirrhosis. This significant reduction in TBK was accompanied by normal serum potassium levels, and slightly decreased serum sodium levels in cirrhosis, however exchangeable sodium (Na-24) was increased in cirrhosis of the liver (55+-13 mmol/kg) compared with controls (40+-7 mmol/kg). These results support the suggestion that changes of sodium-potassium concentration at the cell membrane may regulate the synthesis of Na,K-pump molecules. (orig.) [de

  10. Data in support of FSH induction of IRS-2 in human granulosa cells: Mapping the transcription factor binding sites in human IRS-2 promoter

    Directory of Open Access Journals (Sweden)

    Surleen Kaur

    2016-03-01

    Full Text Available Insulin receptor substrate-2 (IRS-2 plays critical role in the regulation of various metabolic processes by insulin and IGF-1. The defects in its expression and/or function are linked to diseases like polycystic ovary syndrome (PCOS, insulin resistance and cancer. To predict the transcription factors (TFs responsible for the regulation of human IRS-2 gene expression, the transcription factor binding sites (TFBS and the corresponding TFs were investigated by analysis of IRS-2 promoter sequence using MatInspector Genomatix software (Cartharius et al., 2005 [1]. The ibid data is part of author׳s publication (Anjali et al., 2015 [2] that explains Follicle stimulating hormone (FSH mediated IRS-2 promoter activation in human granulosa cells and its importance in the pathophysiology of PCOS. Further analysis was carried out for binary interactions of TF regulatory genes in IRS-2 network using Cytoscape software tool and R-code. In this manuscript, we describe the methodology used for the identification of TFBSs in human IRS-2 promoter region and provide details on experimental procedures, analysis method, validation of data and also the raw files. The purpose of this article is to provide the data on all TFBSs in the promoter region of human IRS-2 gene as it has the potential for prediction of the regulation of IRS-2 gene in normal or diseased cells from patients with metabolic disorders and cancer. Keywords: IRS-2, TFBS, FSH, SP1, ChIP

  11. Ensemble approach combining multiple methods improves human transcription start site prediction

    LENUS (Irish Health Repository)

    Dineen, David G

    2010-11-30

    Abstract Background The computational prediction of transcription start sites is an important unsolved problem. Some recent progress has been made, but many promoters, particularly those not associated with CpG islands, are still difficult to locate using current methods. These methods use different features and training sets, along with a variety of machine learning techniques and result in different prediction sets. Results We demonstrate the heterogeneity of current prediction sets, and take advantage of this heterogeneity to construct a two-level classifier (\\'Profisi Ensemble\\') using predictions from 7 programs, along with 2 other data sources. Support vector machines using \\'full\\' and \\'reduced\\' data sets are combined in an either\\/or approach. We achieve a 14% increase in performance over the current state-of-the-art, as benchmarked by a third-party tool. Conclusions Supervised learning methods are a useful way to combine predictions from diverse sources.

  12. Human calcanei from the Middle Pleistocene site of Sima de los Huesos (Sierra de Atapuerca, Burgos, Spain).

    Science.gov (United States)

    Pablos, Adrián; Martínez, Ignacio; Lorenzo, Carlos; Sala, Nohemi; Gracia-Téllez, Ana; Arsuaga, Juan Luis

    2014-11-01

    The existence of calcanei in the fossil record prior to modern humans and Neandertals is very scarce. This skeletal element is fundamental to understanding the evolution of the morphology of the foot in human evolution. Here we present and metrically and comparatively describe 29 calcaneus remains from the Middle Pleistocene site of Sima de los Huesos (SH) (Sierra de Atapuerca, Burgos, Spain). These calcanei belong to 15 individuals (nine adults, two adolescents and four immature individuals). The metric and morphological differences in the calcanei among Middle and Late Pleistocene hominins tend to be subtle. However, the calcanei from SH are broad and robust with large articular surfaces and most significantly, exhibit a very projected sustentaculum tali. A biomechanical and phylogenetic interpretation is proffered to explain the observed morphology of these calcanei. It has been possible to propose tentative sex assignments for the SH calcanei based on size, using methods similar to those used to establish sex from the talus bones from SH. The estimation of stature based on the calcaneus provides a mean of 175.3 cm for males and 160.6 for females, which is similar to that obtained using other skeletal parts from the site. In sum, the SH calcanei are robust with a proportionally long tubercle and a projected sustentaculum tali, which are traits shared by Neandertals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Polar bear hemoglobin and human Hb A0: same 2,3-diphosphoglycerate binding site but asymmetry of the binding?

    Science.gov (United States)

    Pomponi, Massimo; Bertonati, Claudia; Patamia, Maria; Marta, Maurizio; Derocher, Andrew E; Lydersen, Christian; Kovacs, Kit M; Wiig, Oystein; Bårdgard, Astrid J

    2002-11-01

    Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.

  14. Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans

    International Nuclear Information System (INIS)

    Baron, A.D.; Brechtel, G.; Wallace, P.; Edelman, S.V.

    1988-01-01

    In vivo glucose uptake can occur via two mechanisms, namely, insulin-mediated glucose uptake (IMGU) and non-insulin-mediated glucose uptake (NIMGU). Although the principal tissue sites for IMGU are skeletal muscle, the tissue sites for NIMGU at a given serum glucose concentration are not known. To examine this issue, rates of whole body glucose uptake (Rd) were measured at basal and during glucose clamp studies performed at euglycemia (approximately 90 mg/dl) and hyperglycemia (approximately 220 mg/dl) in six lean healthy men. Studies were performed during hyperinsulinemia (approximately 70 microU/ml) and during somatostatin-induced insulinopenia to measure IMGU and NIMGU, respectively. During each study, leg glucose balance (arteriovenous catheter technique) was also measured. With this approach, rates of whole body skeletal muscle IMGU and NIMGU can be estimated, and the difference between overall Rd and skeletal muscle glucose uptake represents non-skeletal muscle Rd. The results indicate that approximately 20% of basal Rd is into skeletal muscle. During insulinopenia approximately 86% of body NIMGU occurs in non-skeletal muscle tissues at euglycemia. When hyperglycemia was created, whole body NIMGU increased from 128 +/- 6 to 213 +/- 18 mg/min (P less than 0.01); NIMGU into non-skeletal muscle tissues was 134 +/- 11 and 111 +/- 6 mg/min at hyperglycemia and euglycemia, respectively, P = NS. Therefore, virtually all the hyperglycemia induced increment in NIMGU occurred in skeletal muscle. During hyperinsulinemia, IMGU in skeletal muscle represented 75 and 95% of body Rd, at euglycemia and hyperglycemia, respectively

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

    Science.gov (United States)

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

    1997-08-01

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

  16. Evaluation of Human Amniotic Membrane as a Wound Dressing for Split-Thickness Skin-Graft Donor Sites

    Directory of Open Access Journals (Sweden)

    Denys J. Loeffelbein

    2014-01-01

    Full Text Available Human amniotic membrane (HAM has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG donor sites in a swine model (Part A and a clinical trial (Part B. Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU foil (n=8 each. Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker, von Willebrand factor (vWF: angiogenesis, Ki-67 (cell proliferation, and laminin (basement membrane integrity. Part B: STSG donor sites in 45 adult patients (16 female/29 male were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n=15 each. Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative.

  17. Understanding Paleoclimate and Human Evolution Through the Hominin Sites and Paleolakes Drilling Project

    Directory of Open Access Journals (Sweden)

    Kaye Reed

    2009-09-01

    Full Text Available Understanding the evolution of humans and our close relatives is one of the enduring scientific issues of modern times. Since the time of Charles Darwin, scientists have speculated on how and when we evolved and what conditions drove this evolutionary story. The detective work required to address these questions is necessarily interdisciplinary,involving research in anthropology, archaeology, human genetics and genomics, and the earth sciences. In addition to the difficult tasks of finding, describing, and interpreting hominin fossils (the taxonomic tribe which includes Homo sapiens and our close fossil relatives from the last 6 Ma, much of modern geological research associated with paleoanthropology involves understanding the geochronologic and paleoenvironmental context of those fossils. When were they entombed in the sediments? What were the local and regional climatic conditions that early hominins experienced? How did local (watershed scale and regional climate processes combine with regional tectonic boundary conditions to influence hominin food resources, foraging patterns, and demography? How and when did these conditions vary from humid to dry, or cool to warm? Can the history of those conditions (Vrba, 1988; Potts, 1996 be related to the evolution, diversification, stasis, or extinction of hominin species?

  18. The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

    Science.gov (United States)

    Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

    2017-10-01

    A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Characterization of overwintering sites of the invasive brown marmorated stink bug in natural landscapes using human surveyors and detector canines.

    Directory of Open Access Journals (Sweden)

    Doo-Hyung Lee

    Full Text Available Halyomorpha halys is an invasive species from Asia causing major economic losses in agricultural production in the mid-Atlantic region of the United States. Unlike other crop pests, H. halys is also well-known for nuisance problems in urban, suburban, and rural areas, as massive numbers of adults often invade human-made structures to overwinter inside protected environments. Research efforts have focused on populations in human-made structures while overwintering ecology of H. halys in natural landscapes is virtually unknown. We explored forested landscapes in the mid-Atlantic region to locate and characterize natural overwintering structures used by H. halys. We also evaluated the use of detector canines to locate overwintering H. halys to enhance the accuracy and efficiency of surveys. From these studies, we indentified shared characteristics of overwintering sites used by H. halys in natural landscapes. Overwintering H. halys were recovered from dry crevices in dead, standing trees with thick bark, particularly oak (Quercus spp. and locust (Robinia spp.; these characteristics were shared by 11.8% of all dead trees in surveyed landscapes. For trees with favorable characteristics, we sampled ∼20% of the total above-ground tree area and recovered 5.9 adults per tree from the trees with H. halys present. Two detector canines were successfully trained to recognize and detect the odor of adult H. halys yielding >84% accuracy in laboratory and semi-field trials. Detector canines also found overwintering H. halys under field conditions. In particular, overwintering H. halys were recovered only from dead trees that yielded positive indications from the canines and shared key tree characteristics established by human surveyors. The identified characteristics of natural overwintering sites of H. halys will serve as baseline information to establish crop economic risk levels posed by overwintering populations, and accordingly develop sustainable

  20. The two Na+ sites in the human serotonin transporter play distinct roles in the ion coupling and electrogenicity of transport.

    Science.gov (United States)

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H; Henry, L Keith

    2014-01-17

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.

  1. The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport*

    Science.gov (United States)

    Felts, Bruce; Pramod, Akula Bala; Sandtner, Walter; Burbach, Nathan; Bulling, Simon; Sitte, Harald H.; Henry, L. Keith

    2014-01-01

    Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na+, Cl−, and K+ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na+-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca2+ (but not other cations) to functionally replace Na+ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca2+ and Na+ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca2+ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na+-binding sites. PMID:24293367

  2. [(3)H]-YM-09151-2 binding sites in human brain postmortem.

    Science.gov (United States)

    Marazziti, Donatella; Baroni, Stefano; Masala, Irene; Giannaccini, Gino; Betti, Laura; Palego, Lionella; Catena Dell'Osso, Mario; Consoli, Giorgio; Castagna, Maura; Lucacchini, Antonio

    2009-12-01

    The controversial and limited data on the distribution of dopamine (DA) receptors of type 4 (D(4)) in the human brain prompted us to explore their density and pharmacological characteristics in the prefrontal cortex, striatum and hippocampus, through a series of binding assays. Brain samples were taken during autopsy from seven subjects. Tissue homogenates were incubated with increasing concentration of [(3)H]-YM-09151-2, a D(2)-like receptor antagonist, and L-745,870 and/or sulpiride to define the non-specific binding, while PPAP was used to block sigma receptors. The results showed a low density of D(4) receptors in the hippocampus only, with a preponderance of D(2)/D(3) and sigma receptors in the prefrontal cortex and striatum. In conclusion, these findings underline that it is possible to label D(4) receptors by means of [(3)H]-YM-09151-2, provided that D(2), D(3) and sigma receptors are blocked.

  3. Characterization of HSP27 phosphorylation sites in human atherosclerotic plaque secretome

    DEFF Research Database (Denmark)

    Durán, Mari-Carmen; Boeri-Erba, Elisabetta; Mohammed, Shabaz

    2007-01-01

    spectrometry (MS). Among the identified proteins, two isoforms of heat shock protein 27 (HSP27), a protein recently described as a potential biomarker of atherosclerosis, were detected. However, the putative mechanisms in which HSP27 isoforms could be involved in the atherosclerotic process are unknown. Thus......, the role that phosphorylated HSP27 could play in the atherosclerotic process is actually under study. The present work shows the strategies employed to characterize the phosphorylation in the HSP27 secreted by atheroma plaque samples. The application of liquid chromatography tandem mass spectrometry (MS......-lymphocytes). These interactions can be mediated by proteins secreted from these cells, which therefore exert an important role in the atherosclerotic process. We recently described a novel strategy for the characterization of the human atherosclerotic plaque secretome, combining two-dimensional gel electrophoresis and mass...

  4. The evidence for increased L1 activity in the site of human adult brain neurogenesis.

    Directory of Open Access Journals (Sweden)

    Alexey A Kurnosov

    Full Text Available Retroelement activity is a common source of polymorphisms in human genome. The mechanism whereby retroelements contribute to the intraindividual genetic heterogeneity by inserting into the DNA of somatic cells is gaining increasing attention. Brain tissues are suspected to accumulate genetic heterogeneity as a result of the retroelements somatic activity. This study aims to expand our understanding of the role retroelements play in generating somatic mosaicism of neural tissues. Whole-genome Alu and L1 profiling of genomic DNA extracted from the cerebellum, frontal cortex, subventricular zone, dentate gyrus, and the myocardium revealed hundreds of somatic insertions in each of the analyzed tissues. Interestingly, the highest concentration of such insertions was detected in the dentate gyrus-the hotspot of adult neurogenesis. Insertions of retroelements and their activity could produce genetically diverse neuronal subsets, which can be involved in hippocampal-dependent learning and memory.

  5. Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay

    International Nuclear Information System (INIS)

    Crosby, S.R.; Stewart, M.F.; Ratcliffe, J.G.; White, A.

    1988-01-01

    An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful

  6. HC Forum®: a web site based on an international human cytogenetic database

    Science.gov (United States)

    Cohen, Olivier; Mermet, Marie-Ange; Demongeot, Jacques

    2001-01-01

    Familial structural rearrangements of chromosomes represent a factor of malformation risk that could vary over a large range, making genetic counseling difficult. However, they also represent a powerful tool for increasing knowledge of the genome, particularly by studying breakpoints and viable imbalances of the genome. We have developed a collaborative database that now includes data on more than 4100 families, from which we have developed a web site called HC Forum® (http://HCForum.imag.fr). It offers geneticists assistance in diagnosis and in genetic counseling by assessing the malformation risk with statistical models. For researchers, interactive interfaces exhibit the distribution of chromosomal breakpoints and of the genome regions observed at birth in trisomy or in monosomy. Dedicated tools including an interactive pedigree allow electronic submission of data, which will be anonymously shown in a forum for discussions. After validation, data are definitively registered in the database with the email of the sender, allowing direct location of biological material. Thus HC Forum® constitutes a link between diagnosis laboratories and genome research centers, and after 1 year, more than 700 users from about 40 different countries already exist. PMID:11125121

  7. Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase*

    Science.gov (United States)

    Di Nardo, Giovanna; Breitner, Maximilian; Bandino, Andrea; Ghosh, Debashis; Jennings, Gareth K.; Hackett, John C.; Gilardi, Gianfranco

    2015-01-01

    Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp309 in substrate binding and catalysis. These studies predicted an elevated pKa for Asp309 and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp309 with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp309 based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pKa of 8.2 observed for androstenedione binding to the side chain of Asp309. To our knowledge, this work represents the first experimental assignment of a pKa value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures. PMID:25425647

  8. Synthesis and inhibitory evaluation of 3-linked imipramines for the exploration of the S2 site of the human serotonin transporter

    DEFF Research Database (Denmark)

    Brinkø, Anne; Larsen, Maja Thim; Koldsø, Heidi

    2016-01-01

    The human serotonin transporter is the primary target of several antidepressant drugs, and the importance of a primary, high affinity binding site (S1) for antidepressant binding is well documented. The existence of a lower affinity, secondary binding site (S2) has, however, been debated. Herein we...... of the positional relationship between the S1 and S2 sites. The computer simulations suggested that the S2 site does indeed exist although with lower affinity for imipramine than observed within the S1 site. Additionally, it was possible to dock the 3-linked imipramine analogs into positions which occupy the S1...... and the S2 site simultaneously. The structure activity relationship study showed that the shortest ligands were the most potent, and mutations enlarging the proposed S2 site were found to affect the larger ligands positively, while the smaller ligands were mostly unaffected....

  9. Active-Site Environment of Copper-Bound Human Amylin Relevant to Type 2 Diabetes.

    Science.gov (United States)

    Seal, Manas; Dey, Somdatta Ghosh

    2018-01-02

    Type 2 diabetes mellitus (T2Dm) is characterized by reduced β cell mass and amyloid deposits of human islet amyloid polypeptide (hIAPP) or amylin, a 37 amino acid containing peptide around pancreatic β cells. The interaction of copper (Cu) with amylin and its mutants has been studied in detail using absorption, circular dichroism, electron paramagnetic resonance spectroscopy, and cyclic voltammetry. Cu binds amylin in a 1:1 ratio, and the binding domain lies within the first 19 amino acid residues of the peptide. Depending on the pH of the medium, Cu-amylin shows the formation of five pH-dependent components (component IV at pH 4.0, component III at pH 5.0, component II at pH 6.0, component I at pH 8.0, and another higher pH component above pH 9.0). The terminal amine, His18, and amidates are established as key residues in the peptide that coordinate the Cu center. The physiologically relevant components I and II can generate H 2 O 2 , which can possibly account for the enhanced toxicity of amylin in the presence of Cu, causing damage of the β cells of the pancreas via oxidative stress.

  10. Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

    Science.gov (United States)

    Wise, John G

    2012-06-26

    Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

  11. Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

    Science.gov (United States)

    Salter, D M; Godolphin, J L; Gourlay, M S

    1995-04-01

    During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

  12. The presence of Fasciola hepatica (Liver-fluke in humans and cattle from a 4,500 Year old archaeological site in the Saale-Unstrut Valley, Germany

    Directory of Open Access Journals (Sweden)

    Dittmar K

    2003-01-01

    Full Text Available During an excavation of a site of the corded ware culture in the Saale-Unstrut-Valley (ca. 3000 BC in Germany, a soil sample from the pelvis of a human skeleton was studied under palaeoparasitological aspects. Eggs of the trematode Fasciola hepatica and of the nematode genus Capillaria were found. This is the first case of a direct association of a F. hepatica-infestation to both a prehistoric human skeleton and domesticated animal remains. Sheep and cattle bones were present at the same site and F. hepatica eggs were found in bovine samples. This strongly points toward an existing infection cycle, involving humans as a final host.

  13. Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

    Science.gov (United States)

    Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

    1994-07-01

    We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

  14. The Hominin Sites and Paleolakes Drilling Project (HSPDP): Understanding the paleoenvironmental and paleoclimatic context of human origins through continental drilling

    Science.gov (United States)

    Cohen, Andrew S.; Campisano, Christopher; Asrat, Asfawossen; Arrowsmith, Ramon; Deino, Alan; Feibel, Craig; Hill, Andrew; Kingston, John; Lamb, Henry; Lowenstein, Tim; Olago, Daniel; Bernhart Owen, R.; Renaut, Robin; Schabitz, Frank; Trauth, Martin

    2015-04-01

    The influence of climate and environmental history on human evolution is an existential question that continues to be hotly debated, in part because of the paucity of high resolution records collected in close proximity to the key fossil and archaeological evidence. To address this issue and transform the scientific debate, the HSPDP was developed to collect lacustrine sediment drill cores from basins in Kenya and Ethiopia that collectively encompass critical time intervals and locations for Plio-Quaternary human evolution in East Africa. After a 17 month campaign, drilling was completed in November, 2014, with over 1750m of core collected from 11 boreholes from five areas (1930m total drilling length, avg. 91% recovery). The sites, from oldest to youngest, include 1) N. Awash, Ethiopia (~3.5-2.9Ma core interval); 2) Baringo-Tugen Hills, Kenya (~3.3-2.5Ma); 3) West Turkana, Kenya (~1.9-1.4Ma); L. Magadi, Kenya (0.8-0Ma) and the Chew Bahir Basin, Ethiopia (~0.5-0Ma). Initial core description (ICD) and sampling for geochronology, geochemistry and paleoecology studies had been completed by mid2014, with the two remaining sites (Magadi and Chew Bahir) scheduled for ICD work in early 2015. Whereas the primary scientific targets were the lacustrine deposits from the hominin-bearing basin depocenters, many intervals of paleosols (representative of low lake stands and probable arid periods) were also encountered in drill cores. Preliminary analyses of drill core sedimentology and geochemistry show both long-term lake level changes and cyclic variability in lake levels, both of which may be indicative of climatic forcing events of interest to paleoanthropologists. Authors of this abstract also include the entire HSPDP field team.

  15. Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

    Science.gov (United States)

    Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal Soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

    2014-09-01

    Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

  16. Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

    Directory of Open Access Journals (Sweden)

    Tshidi Tsibane

    Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

  17. Human talus bones from the Middle Pleistocene site of Sima de los Huesos (Sierra de Atapuerca, Burgos, Spain).

    Science.gov (United States)

    Pablos, Adrián; Martínez, Ignacio; Lorenzo, Carlos; Gracia, Ana; Sala, Nohemi; Arsuaga, Juan Luis

    2013-07-01

    Here we present and describe comparatively 25 talus bones from the Middle Pleistocene site of the Sima de los Huesos (SH) (Sierra de Atapuerca, Burgos, Spain). These tali belong to 14 individuals (11 adult and three immature). Although variation among Middle and Late Pleistocene tali tends to be subtle, this study has identified unique morphological characteristics of the SH tali. They are vertically shorter than those of Late Pleistocene Homo sapiens, and show a shorter head and a broader lateral malleolar facet than all of the samples. Moreover, a few shared characters with Neanderthals are consistent with the hypothesis that the SH population and Neanderthals are sister groups. These shared characters are a broad lateral malleolar facet, a trochlear height intermediate between modern humans and Late Pleistocene H. sapiens, and a short middle calcaneal facet. It has been possible to propose sex assignment for the SH tali based on their size. Stature estimates based on these fossils give a mean stature of 174.4 cm for males and 161.9 cm for females, similar to that obtained based on the long bones from this same site. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Attachment sites of the coracoclavicular ligaments are characterized by fibrocartilage differentiation: a study on human cadaveric tissue.

    Science.gov (United States)

    Ockert, B; Braunstein, V; Sprecher, C; Shinohara, Y; Kirchhoff, C; Milz, S

    2012-02-01

    We analyzed the immunohistochemical labeling patterns of the extracellular matrix of the coracoclavicular ligaments (CCL) in order to relate the molecular composition of the attachment sites to their mechanical environment. Ligaments were exposed from 12 fresh-frozen human cadaveric samples (four males, mean age: 48.6 ± 12.1 years). Cryosection of methanol-fixed and decalcified tissue was cut and sections were labeled with a panel of monoclonal antibodies directed against collagens, proteoglycans and proteins of vascular components. Attachment sites of both ligaments showed characteristic fibrocartilaginous labeling of collagen type II, aggrecan and link protein in all samples. Labeling for type II collagen was most conspicuous at the insertion of the coracoid process. Morphometry of adjacent samples revealed a fibrocartilage zone of 10-15% in relationship with the ligament proper, where labeling for type II collagen, aggrecan and link protein was negative. The presence of fibrocartilage at both entheses of the trapezoid and conoid ligament suggests that the CCL complex is subject to shear/compression forces. A variable fibrocartilage differentiation at the entheses of both ligaments may be related to the marked change in loading and insertion angle that the ligaments undergo during shoulder movement. © 2010 John Wiley & Sons A/S.

  19. Exploring the site-selective binding of jatrorrhizine to human serum albumin: spectroscopic and molecular modeling approaches.

    Science.gov (United States)

    Mi, Ran; Hu, Yan-Jun; Fan, Xiao-Yang; Ouyang, Yu; Bai, Ai-Min

    2014-01-03

    This paper exploring the site-selective binding of jatrorrhizine to human serum albumin (HSA) under physiological conditions (pH=7.4). The investigation was carried out using fluorescence spectroscopy, UV-vis spectroscopy, and molecular modeling. The results of fluorescence quenching and UV-vis absorption spectra experiments indicated the formation of the complex of HSA-jatrorrhizine. Binding parameters calculating from Stern-Volmer method and Scatchard method were calculated at 298, 304 and 310 K, with the corresponding thermodynamic parameters ΔG, ΔH and ΔS as well. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that jatrorrhizine bind to HSA with the binding affinities of the order 10(4) L mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for jatrorrhizine-HSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that jatrorrhizine is mainly located within the hydrophobic pocket of the subdomain IIIA of HSA. Furthermore, the synchronous fluorescence spectra suggested that the association between jatrorrhizine and HSA changed molecular conformation of HSA. Copyright © 2013. Published by Elsevier B.V.

  20. Probing of possible olanzapine binding site on human serum albumin: Combination of spectroscopic methods and molecular dynamics simulation

    International Nuclear Information System (INIS)

    Shahlaei, Mohsen; Rahimi, Behnoosh; Ashrafi-Kooshk, Mohammad Reza; Sadrjavadi, Komail; Khodarahmi, Reza

    2015-01-01

    Human serum albumin (HSA)-drug binding affinity is one of the major factors that determine the pharmacokinetics, halftime and bioavailability of drugs in various tissues. In the present study, the interaction of olanzapine (OLZ), a thienobenzodiazepine drug, administered for the treatment of schizophrenia and bipolar disorder, with HSA has been studied using spectroscopic methods such as ultraviolet absorbance, fluorescence and FTIR combined with computational procedures. Analyzing of the Stern–Volmer quenching data showed only one primary binding site on HSA with a binding constant of 4.12×10 4 M −1 at 298 K. Thermodynamic analyses showed enthalpy change (ΔH°) and entropy change (ΔS°) were 28.03±3.42 kJ mol −1 and −25.52±11.52 J mol −1 K −1 , respectively. Molecular docking results suggested the hydrophobic residues such as Val 216 , Leu 327 , Ala 350 and polar residues such as Glu 354 play an important role in the drug binding. Decrement in α-helix content of the protein upon OLZ binding was also confirmed by evidences provided by molecular dynamics simulation as well as FTIR spectroscopy. - Highlights: • Leu 327 , Ala 350 as well as hydrophilic residues of HSA play an important role in the binding reaction. • The drug has only one primary binding site on HSA with a binding constant of 4.12×10 4 M −1 at 298 K. • The drug binds near to site I

  1. Probing of possible olanzapine binding site on human serum albumin: Combination of spectroscopic methods and molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Shahlaei, Mohsen, E-mail: mohsenshahlaei@yahoo.com [Nano drug delivery research Center, Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Rahimi, Behnoosh [Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Student research committee, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Sadrjavadi, Komail [Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2015-02-15

    Human serum albumin (HSA)-drug binding affinity is one of the major factors that determine the pharmacokinetics, halftime and bioavailability of drugs in various tissues. In the present study, the interaction of olanzapine (OLZ), a thienobenzodiazepine drug, administered for the treatment of schizophrenia and bipolar disorder, with HSA has been studied using spectroscopic methods such as ultraviolet absorbance, fluorescence and FTIR combined with computational procedures. Analyzing of the Stern–Volmer quenching data showed only one primary binding site on HSA with a binding constant of 4.12×10{sup 4} M{sup −1} at 298 K. Thermodynamic analyses showed enthalpy change (ΔH°) and entropy change (ΔS°) were 28.03±3.42 kJ mol{sup −1} and −25.52±11.52 J mol{sup −1} K{sup −1}, respectively. Molecular docking results suggested the hydrophobic residues such as Val{sub 216}, Leu{sub 327}, Ala{sub 350} and polar residues such as Glu{sub 354} play an important role in the drug binding. Decrement in α-helix content of the protein upon OLZ binding was also confirmed by evidences provided by molecular dynamics simulation as well as FTIR spectroscopy. - Highlights: • Leu{sub 327}, Ala{sub 350} as well as hydrophilic residues of HSA play an important role in the binding reaction. • The drug has only one primary binding site on HSA with a binding constant of 4.12×10{sup 4} M{sup −1} at 298 K. • The drug binds near to site I.

  2. Early Pleistocene human hand phalanx from the Sima del Elefante (TE) cave site in Sierra de Atapuerca (Spain).

    Science.gov (United States)

    Lorenzo, Carlos; Pablos, Adrián; Carretero, José Miguel; Huguet, Rosa; Valverdú, Josep; Martinón-Torres, María; Arsuaga, Juan Luis; Carbonell, Eudald; Bermúdez de Castro, José María

    2015-01-01

    In this study, a new Early Pleistocene proximal hand phalanx (ATE9-2) from the Sima del Elefante cave site (TE - Sierra de Atapuerca, Spain), ascribed to Homo sp., is presented and comparatively described in the context of the evolution of the genus Homo. The ATE9-2 specimen is especially important because of the paucity of hand bones in the human fossil record during the Early Pleistocene. The morphological and metrical analyses of the phalanx ATE9-2 indicate that there are no essential differences between it and comparator fossil specimens for the genus Homo after 1.3 Ma (millions of years ago). Similar to Sima de los Huesos and Neandertal specimens, ATE9-2 is a robust proximal hand phalanx, probably reflecting greater overall body robusticity in these populations or a higher gracility in modern humans. The age of level TE9 from Sima del Elefante and morphological and metrical studies of ATE9-2 suggest that the morphology of the proximal hand phalanges and, thus, the morphology of the hand could have remained stable over the last 1.2-1.3 Ma. Taking into account the evidence recently provided by a metacarpal from Kaitio (Kenya) from around 1.42 Ma, we argue that modern hand morphology is present in the genus Homo subsequent to Homo habilis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Anionic Sites, Fucose Residues and Class I Human Leukocyte Antigen Fate During Interaction of Toxoplasma gondii with Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Stumbo Ana Carolina

    2002-01-01

    Full Text Available Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles.

  4. Ecotoxicity and risk to human fish consumers of polychlorinated biphenyls in fish near the Hanford Site (USA).

    Science.gov (United States)

    Delistraty, Damon

    2013-02-15

    The purpose of this study was to quantify three groups of polychlorinated biphenyl (PCB) congeners (i.e., dioxin-like toxic equivalents [TEQ], non-dioxin-like PCBs, total PCBs) in fish in several species, tissues, and locations in the Columbia River near the Hanford Site. For TEQ and total PCBs, fish ecotoxicity and risk to human fish consumers were also evaluated. Non-dioxin-like PCBs were not assessed for toxicity, due to lack of available benchmarks. In sturgeon liver, TEQ was significantly higher (Pfillet than in other species (except carp) and significantly higher (Pfillet, relative to bass. All PCB residues in carcass were significantly elevated (Pfillet. In addition to PCB source, many factors (e.g., dietary composition, tissue lipid content, fish mobility and home range, age, toxicokinetic processes, seasonal adaptations) influence patterns in PCB bioaccumulation across species, tissues, and locations. TEQ and total PCB residues in liver, fillet, and carcass, observed in this study, were below corresponding no effect residues for TEQ and Aroclors in the literature for fish survival, growth, and reproduction. In contrast, TEQ and total PCBs in fillet in this study exceeded USEPA tissue screening levels for cancer (1E-6 risk) and noncancer (hazard quotient [HQ]=1) toxicity for human fish consumers. Key uncertainties in these comparisons to assess toxicity relate to variation in fish species sensitivity to PCBs and use of Aroclor data in the literature to represent total PCBs. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

    Science.gov (United States)

    Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

    2014-04-25

    The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Surgical Site Infection after Sternotomy in Low- and Middle-Human Development Index Countries: A Systematic Review.

    Science.gov (United States)

    Forrester, Joseph D; Cai, Lawrence Z; Zeigler, Sanford; Weiser, Thomas G

    2017-10-01

    The burden of cardiovascular disease is increasing in low- and middle-human development index (LMHDI) countries, and cardiac operations are an important component of a comprehensive cardiovascular care package. Little is known about the baseline incidence of surgical site infections (SSIs) among patients undergoing sternotomy in LMHDI countries. A prospectively registered, systematic literature review of articles in the PubMed, Ovid, and Web of Science databases describing the epidemiology and management of SSIs among persons undergoing sternotomy in LMHDI countries was performed. We performed a quantitative synthesis of patients undergoing sternotomy for CABG to estimate published sternotomy SSI rates. Of the 423 abstracts identified after applying search criteria, 14 studies were reviewed in detail. The pooled SSI rate after sternotomy among reviewed studies was 4.3 infections per 100 sternotomies (95% confidence interval [CI] 1.3-6.0 infections per 100 sternotomies), which is comparable to infection rates in high-human development index countries. As the burden of cardiovascular disease in LMHDI settings increases, the ability to provide safe cardiac surgical care is paramount. Describing the baseline SSI rate after sternotomy in LMHDI countries is an important first step in creating baseline expectations for SSI rates in cardiac surgical programs in these settings.

  7. Localisation and quantification of alkali-labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Cerda-Flores, R M; Fernández, J L; López-Fernández, C; Aragón Tovar, A R; Gosálvez, J

    2015-03-01

    The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility. © 2014 Blackwell Verlag GmbH.

  8. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    Directory of Open Access Journals (Sweden)

    Cédric Grauffel

    Full Text Available Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs. In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p. To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG, hNaa10p/AcCoA/substrate (substrate=MLG, EEE. Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic, hNaa20p (hydrophobic/basic, hNaa30p (basic and hNaa50p (hydrophobic. We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide

  9. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    Science.gov (United States)

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  10. Middle Pleistocene lower back and pelvis from an aged human individual from the Sima de los Huesos site, Spain.

    Science.gov (United States)

    Bonmatí, Alejandro; Gómez-Olivencia, Asier; Arsuaga, Juan-Luis; Carretero, José Miguel; Gracia, Ana; Martínez, Ignacio; Lorenzo, Carlos; Bérmudez de Castro, José María; Carbonell, Eudald

    2010-10-26

    We report a nearly complete lumbar spine from the Middle Pleistocene site of the Sima de los Huesos (SH) that is assigned to the previously published SH male Pelvis 1 [Arsuaga JL, et al. (1999). Nature 399: 255-258]. The "SH Pelvis 1 individual" is a unique nearly complete lumbo-pelvic complex from the human Middle Pleistocene fossil record, and offers a rare glimpse into the anatomy and past lifeways of Homo heidelbergensis. A revised reconstruction of Pelvis 1, together with the current fossil evidence, confirms our previous hypothesis that the morphology of this pelvis represents the primitive pattern within the genus Homo. Here we argue that this primitive pattern is also characterized by sexual dimorphism in the pelvic canal shape, implying complicated deliveries. In addition, this individual shows signs of lumbar kyphotic deformity, spondylolisthesis, and Baastrup disease. This suite of lesions would have postural consequences and was most likely painful. As a result, the individual's daily physical activities would have been restricted to some extent. Reexamination of the age-at-death agrees with this individual being over 45 y old, relying on the modern human pattern of changes of the articular surfaces of the os coxae. The presence of degenerative pathological lesions and the advanced age-at-death of this individual make it the most ancient postcranial evidence of an aged individual in the human fossil record. Additional nonpathological SH lumbo-pelvic remains are consistent with previous hypotheses, suggesting a less-pronounced sagittal spinal curvature in Neandertals compared with Homo sapiens.

  11. Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

    Science.gov (United States)

    Muraki, Michiro; Hirota, Kiyonori

    2017-07-03

    Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem. A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain. The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

  12. The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells.

    Science.gov (United States)

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-04-01

    The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

  13. A common-sense probabilistic approach to assessing inadvertent human intrusion into low-level radioactive waste at the Nevada Test Site

    International Nuclear Information System (INIS)

    Black, P.; Hooten, M.; Black, K.; Moore, B.; Rawlinson, S.; Barker, L.

    1997-01-01

    Each site disposing of low-level radioactive waste is required to prepare and maintain a site-specific performance assessment (1) to determine potential risks posed by waste management systems to the public, and the environment, and (2) to compare these risks to established performance objectives. The DOE Nevada Operations Office, Waste Management Program recently completed a one-year study of site-specific scenarios for inadvertent human intrusion by drilling into buried low-level radioactive waste sites, as part of ongoing performance assessment studies. Intrusion scenarios focus on possible penetration of buried waste through drilling for sources of groundwater. The probability of drilling penetration into waste was judged to be driven primarily by two settlement scenarios: (1) scattered individual homesteaders, and (2) a community scenario consisting of a cluster of settlers that share drilling and distribution systems for groundwater. Management control factors include institutional control, site knowledge, placards and markers, surface barriers, and subsurface barriers. The Subject Matter Experts concluded that institutional control and site knowledge may be important factors for the first few centuries, but are not significant over the evaluation period of 10,000 years. Surface barriers can be designed that would deter the siting of a drill rig over the waste site to an effectiveness of 95%. Subsurface barriers and placards and markers will not as effectively prevent inadvertent human intrusion. Homestead and community scenarios were considered by the panel to render a site-specific probability of around 10% for inadvertent human intrusion. If management controls are designed and implemented effectively, then the probability of inadvertent human intrusion can be reduced to less than 1%

  14. A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

    Science.gov (United States)

    Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

    2018-01-01

    Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

  15. Human cytomegalovirus-encoded miR-US4-1 promotes cell ...

    Indian Academy of Sciences (India)

    2016-04-05

    Apr 5, 2016 ... 3′) for hcmv-miR-US4-1 was designed according to the. miRNA sequence (5′- ... the hcmv-miR-US4-1 hybrid primer and polyA tails were. 184. Yaozhong ... eight hours later, luciferase activities were measured us- ing Dual ..... resolution profiling and analysis of viral and host small RNAs during human ...

  16. On-site and off-site atmospheric PBDEs in an electronic dismantling workshop in south China: Gas-particle partitioning and human exposure assessment

    International Nuclear Information System (INIS)

    An Taicheng; Zhang Delin; Li Guiying; Mai Bixian; Fu Jiamo

    2011-01-01

    Gas samples and total suspended particle during work and off work time were investigated on-site and off-site electronic waste dismantling workshop (I- and O-EWDW), then compared with plastic recycling workshop (PRW) and waste incineration plant (WIP). TSP concentrations and total PBDE were 0.36-2.21 mg/m 3 and 27-2975 ng/m 3 at different workshops, respectively. BDE-47, -99, and -209 were major ΣPBDE congeners at I-EWDW and WIP, while BDE-209 was only dominant congener in PRW and control sites during work time and all sites during off work time. The gas-particle partitioning result was well correlated with the subcooled liquid vapor pressure for all samples, except for WIP and I-EDWD, at park during work time, and residential area during off work time. The predicted urban curve fitted well with measured φ values at O-DEWD during work time, whereas it was slightly overestimated or underestimated for others. Exposure assessment revealed the highest exposure site was I-EDWD. - Highlights: → On- and off-site atmospheric PBDEs was monitored in e-waste dismantling workshops in south China. → The gas-particle partitioning result was well correlated with the subcooled liquid vapor pressure for some samples. → Exposure assessment revealed that workers in I-EDWD were the highest exposure population. - The findings of this study may serve as a valuable reference for future risk assessment and environmental management in Guiyu, South China.

  17. A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells.

    Science.gov (United States)

    Sato, Takashi; Watanabe, Mami; Hashimoto, Kei; Ota, Tomoko; Akimoto, Noriko; Imada, Keisuke; Nomizu, Motoyoshi; Ito, Akira

    2012-01-01

    EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of

  18. Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

    1986-01-01

    Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

  19. Substitution of Active Site Tyrosines with Tryptophan Alters the Free Energy for Nucleotide Flipping by Human Alkyladenine DNA Glycosylase†

    Science.gov (United States)

    Hendershot, Jenna M.; Wolfe, Abigail E.; O'Brien, Patrick J.

    2011-01-01

    Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N6-ethenoadenine (εA), within 4-fold of that of the wildtype enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40 to 100-fold decreases in the flipping equilibrium relative to wildtype. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in

  20. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

    DEFF Research Database (Denmark)

    Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming

    2012-01-01

    a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most......RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed...... changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential...

  1. The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

    Science.gov (United States)

    Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

    2017-06-01

    Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

  2. What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

    Science.gov (United States)

    Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

    2008-08-01

    Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

  3. Landscapes, depositional environments and human occupation at Middle Paleolithic open-air sites in the southern Levant, with new insights from Nesher Ramla, Israel

    Science.gov (United States)

    Zaidner, Yossi; Frumkin, Amos; Friesem, David; Tsatskin, Alexander; Shahack-Gross, Ruth

    2016-04-01

    Middle Paleolithic human occupation in the Levant (250-50 ka ago) has been recorded in roofed (cave and rockshelter) and open-air sites. Research at these different types of sites yielded different perspectives on the Middle Paleolithic human behavior and evolution. Until recently, open-air Middle Paleolithic sites in the Levant were found in three major sedimentary environments: fluvial, lake-margin and spring. Here we describe a unique depositional environment and formation processes at the recently discovered open-air site of Nesher Ramla (Israel) and discuss their contribution to understanding site formation processes in open-air sites in the Levant. The site is 8-m-thick Middle Paleolithic sequence (OSL dated to 170-80 ka) that is located in a karst sinkhole formed by gravitational deformation and sagging into underground voids. The sedimentary sequence was shaped by gravitational collapse, cyclic colluviation of soil and gravel into the depression, waterlogging, in situ pedogenesis and human occupation. Original bedding and combustion features are well-preserved in the Lower archaeological sequence, a rare occurrence in comparison to other open-air archaeological sites. This phenomenon coincides with episodes of fast sedimentation/burial, which also allowed better preservation of microscopic remains such as ash. The Upper archaeological sequence does not exhibit bedding or preservation of ash, despite presence of heat-affected lithic artifacts, which makes it similar to other open-air sites in the Levant. We suggest that rate of burial is the major factor that caused the difference between the Upper and Lower sequences. The differences in the burial rate may be connected to environmental and vegetation changes at the end of MIS 6. We also identified an interplay between sediment in-wash and density of human activity remains, i.e. during episodes of low natural sediment input the density of artifacts is higher relative to episodes with high rate of sediment in

  4. Tracking Human-Induced Landscape Disturbance at the Nasca Lines UNESCO World Heritage Site in Peru with COSMO-SkyMed InSAR

    OpenAIRE

    Francesca Cigna; Deodato Tapete

    2018-01-01

    The “Lines and Geoglyphs of Nasca and Palpa” in Peru are among the most well-known UNESCO World Heritage Sites globally, and an exemplar of site where heritage assets cannot be separated from their natural and anthropogenic environment. The site is exposed to interactions with natural processes, as well as human presence. In this work, 3-m resolution synthetic aperture radar (SAR) StripMap HIMAGE HH-polarised scenes acquired by the X-band COSMO-SkyMed constellation are exploited to track two ...

  5. Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

    International Nuclear Information System (INIS)

    Funk, W.D.; MacGillivray, R.T.A.; Mason, A.B.; Brown, S.A.; Woodworth, R.C.

    1990-01-01

    A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO 4 -polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19 F-Tyr hTF/2N gave four well-resolved 19 F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

  6. Induction of human interferon gene expression is associated with a nuclear factor that interacts with the site of the human immunodeficiency virus-enhancer

    International Nuclear Information System (INIS)

    Hiscott, J.; Alper, D.; Cohen, L.; Leblanc, J.F.; Sportza, L.; Wong, A.; Xanthoudakis, S.

    1989-01-01

    The relationship between transcription of alpha and beta interferon (IFN-α and IFN-β) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-α2 (rIFN-α2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-α2 prior to Sendai virus infection increased the mRNA levels of IFN-α1, IFN-α2, and IFN-β as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-α1 and IFN-β regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-α1 and IFN-β promoters appear to be distinct DNA-binding proteins. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-β regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-κB-like site within the IFN-β promoter

  7. Ochres from rituals of prehistoric human funerals at the Toca do Enoque site, Piauí, Brazil

    Science.gov (United States)

    Cavalcante, Luis Carlos Duarte; da Luz, Maria De Fátima; Guidon, Niéde; Fabris, José Domingos; Ardisson, José Domingos

    2011-11-01

    The archaeological site known as Toca do Enoque (geographical coordinates, 09° 14' 65.3″ S 43° 55' 62.5″ W) is a rock shelter located in the Serra das Andorinhas (Serra das Confusões National Park), rural area of the city of Guaribas, state of Piauí, Brazil. Several rupestrian paintings (anthropomorphic and zoomorphic motifs along with some pure graphisms), predominantly in red, are found on the sandstone walls. Charcoals, lithic materials, necklaces with teeth, animal bones, gastropod shells, ochres and human skeletons (dated from 6,220 ± 40 to 6,610 ± 40 years before present, BP) were identified in recent excavations in this shelter. Red and yellow ochre samples were collected from prehistoric funeral structures and analyzed with powder X-ray diffractometry, Fourier-transform infrared spectroscopy and 57Fe transmission Mössbauer spectroscopy at 298 K and 80 K. Mössbauer data indicate that the red ochre do contain predominantly hematite ( α-Fe2O3) whereas goethite ( α-FeOOH) is the major mineral in the yellow ochre.

  8. Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

    Directory of Open Access Journals (Sweden)

    Caroline Y. Kuo

    2018-05-01

    Full Text Available X-linked hyper-immunoglobulin M (hyper-IgM syndrome (XHIM is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9 platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs, as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

  9. Comparative Genomic Hybridization of Human Malignant Gliomas Reveals Multiple Amplification Sites and Nonrandom Chromosomal Gains and Losses

    Science.gov (United States)

    Schròck, Evelin; Thiel, Gundula; Lozanova, Tanka; du Manoir, Stanislas; Meffert, Marie-Christine; Jauch, Anna; Speicher, Michael R.; Nürnberg, Peter; Vogel, Siegfried; Janisch, Werner; Donis-Keller, Helen; Ried, Thomas; Witkowski, Regine; Cremer, Thomas

    1994-01-01

    Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue ImagesFigure 1Figure 4Figure 6 PMID:8203461

  10. Environmental contamination and human exposure to dioxin-related compounds in e-waste recycling sites of developing countries.

    Science.gov (United States)

    Tue, Nguyen Minh; Takahashi, Shin; Subramanian, Annamalai; Sakai, Shinichi; Tanabe, Shinsuke

    2013-07-01

    E-waste recycling using uncontrolled processes is a major source of dioxin-related compounds (DRCs), including not only the regulated polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) but also non-regulated brominated and mixed halogenated compounds (PBDD/Fs and PXDD/Fs). Various studies at informal e-waste recycling sites (EWRSs) in Asian developing countries found the soil contamination levels of PCDD/Fs from tens to ten thousand picogram TCDD-equivalents (TEQ) per gram and those of DL-PCBs up to hundreds of picogram TEQ per gram. The air concentration of PCDD/Fs was reported as high as 50 pg TEQ per m(3) in Guiyu, the largest Chinese EWRS. Non-regulated compounds also contributed substantially to the total DL toxicity of the DRC mixtures from e-waste, as evidenced by the high TEQ levels estimated for the currently identifiable PBDD/Fs as well as the large portion of unexplained bioassay-derived TEQ levels in soils/dusts from EWRSs. Considering the high exposure levels estimated for EWRS residents, especially children, comprehensive emission inventories of DRCs from informal e-waste recycling, the identities and toxic potencies of unidentified DRCs released, and their impacts on human health need to be investigated in future studies.

  11. Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3' end.

    Science.gov (United States)

    Yoshida, Tetsuya; Kitazawa, Yugo; Komatsu, Ken; Neriya, Yutaro; Ishikawa, Kazuya; Fujita, Naoko; Hashimoto, Masayoshi; Maejima, Kensaku; Yamaji, Yasuyuki; Namba, Shigetou

    2014-11-01

    In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3'-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6-68.7, 49.9-70.8, 31.0-70.8 and 39.4-70.1 %, respectively, at the nucleotide level and 39.8-75.0, 43.6-77.8, 19.2-70.4 and 31.2-74.2 %, respectively, at the amino acid level. The 5'- and 3'-UTRs of HLFPV-J showed 24.3-58.6 and 13.0-79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.

  12. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export*

    Science.gov (United States)

    Port, Sarah A.; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H.

    2016-01-01

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. PMID:27613868

  13. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    Science.gov (United States)

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. The thyroxine-binding site of human apolipoprotein-A-I: Location in the N-terminal domain

    International Nuclear Information System (INIS)

    Benvenga, S.; Cahnmann, H.J.; Robbins, J.

    1991-01-01

    We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [125I]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [125I]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7,10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 greater than MAb 14 greater than MAb 4 greater than MAb 18. In the case of lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipid-complexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the N-terminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site

  15. Ecological Wisdom and Inspiration Underlying the Planning and Construction of Ancient Human Settlements: Case Study of Hongcun UNESCO World Heritage Site in China

    Directory of Open Access Journals (Sweden)

    Shanwen Zheng

    2018-04-01

    Full Text Available Human settlements are social-economic-natural complex ecosystems centered on human activities and the most prominent site for the contradictions between humans and the environment. Taking Hongcun, a UNESCO World Heritage site in China, as an example, this paper analyzes the methods and effect of coupling man and nature in Hongcun, summarizes the ecological wisdom of dealing with the relationship between human and nature, and uses this wisdom to shed light on the planning, construction, and management of contemporary urban and rural settlements. Firstly, the study introduces the Human-Natural Intergraded Ecological Planning (HNIEP model’s hypothesis, explaining its foundation and potential principles or approaches. Secondly, using the case study of Hongcun to explain, support, and validate the HNIEP model and its framework, the study found that the unique planning and construction of Hongcun has greatly promoted ecosystem services, such as local microclimate regulation, rainwater runoff regulation, water conservation, landscape aesthetic, and engagement with nature. Thirdly, Hongcun reflects the concept of harmonious coexistence between human and nature, the wisdom of rational use of ecosystem structures, processes and functions, and the wisdom of coupling human activities with the living environment and natural ecosystem. Finally, the paper summarizes the enlightenment brought by both the HNIEP model and Hongcun wisdom to contemporary urban-rural planning and construction management.

  16. Metal uptake by homegrown vegetables – The relative importance in human health risk assessments at contaminated sites

    Energy Technology Data Exchange (ETDEWEB)

    Augustsson, Anna L.M., E-mail: anna.augustsson@lnu.se [Department of Biology and Environmental Science, Linnaeus University, SE-391 82 Kalmar (Sweden); Uddh-Söderberg, Terese E. [Department of Biology and Environmental Science, Linnaeus University, SE-391 82 Kalmar (Sweden); Hogmalm, K. Johan [Department of Earth Sciences, University of Gothenburg, Gothenburg (Sweden); Filipsson, Monika E.M. [Department of Biology and Environmental Science, Linnaeus University, SE-391 82 Kalmar (Sweden)

    2015-04-15

    Risk assessments of contaminated land often involve the use of generic bioconcentration factors (BCFs), which express contaminant concentrations in edible plant parts as a function of the concentration in soil, in order to assess the risks associated with consumption of homegrown vegetables. This study aimed to quantify variability in BCFs and evaluate the implications of this variability for human exposure assessments, focusing on cadmium (Cd) and lead (Pb) in lettuce and potatoes sampled around 22 contaminated glassworks sites. In addition, risks associated with measured Cd and Pb concentrations in soil and vegetable samples were characterized and a probabilistic exposure assessment was conducted to estimate the likelihood of local residents exceeding tolerable daily intakes. The results show that concentrations in vegetables were only moderately elevated despite high concentrations in soil, and most samples complied with applicable foodstuff legislation. Still, the daily intake of Cd (but not Pb) was assessed to exceed toxicological thresholds for about a fifth of the study population. Bioconcentration factors were found to vary more than indicated by previous studies, but decreasing BCFs with increasing metal concentrations in the soil can explain why the calculated exposure is only moderately affected by the choice of BCF value when generic soil guideline values are exceeded and the risk may be unacceptable. - Highlights: • Uptake of Cd and Pb by lettuce and potatoes increased with soil contamination. • Consumption of homegrown vegetables may lead to a daily Cd intake above TDIs. • The variability in the calculated BCFs is high when compared to previous studies. • Exposure assessments are most sensitive to the choice of BCFs at low contamination.

  17. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  18. Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

    Science.gov (United States)

    Keough, D T; Emmerson, B T; de Jersey, J

    1991-02-22

    Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

  19. Metal uptake by homegrown vegetables – The relative importance in human health risk assessments at contaminated sites

    International Nuclear Information System (INIS)

    Augustsson, Anna L.M.; Uddh-Söderberg, Terese E.; Hogmalm, K. Johan; Filipsson, Monika E.M.

    2015-01-01

    Risk assessments of contaminated land often involve the use of generic bioconcentration factors (BCFs), which express contaminant concentrations in edible plant parts as a function of the concentration in soil, in order to assess the risks associated with consumption of homegrown vegetables. This study aimed to quantify variability in BCFs and evaluate the implications of this variability for human exposure assessments, focusing on cadmium (Cd) and lead (Pb) in lettuce and potatoes sampled around 22 contaminated glassworks sites. In addition, risks associated with measured Cd and Pb concentrations in soil and vegetable samples were characterized and a probabilistic exposure assessment was conducted to estimate the likelihood of local residents exceeding tolerable daily intakes. The results show that concentrations in vegetables were only moderately elevated despite high concentrations in soil, and most samples complied with applicable foodstuff legislation. Still, the daily intake of Cd (but not Pb) was assessed to exceed toxicological thresholds for about a fifth of the study population. Bioconcentration factors were found to vary more than indicated by previous studies, but decreasing BCFs with increasing metal concentrations in the soil can explain why the calculated exposure is only moderately affected by the choice of BCF value when generic soil guideline values are exceeded and the risk may be unacceptable. - Highlights: • Uptake of Cd and Pb by lettuce and potatoes increased with soil contamination. • Consumption of homegrown vegetables may lead to a daily Cd intake above TDIs. • The variability in the calculated BCFs is high when compared to previous studies. • Exposure assessments are most sensitive to the choice of BCFs at low contamination

  20. Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

    KAUST Repository

    Reymer, A.

    2009-07-08

    To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.

  1. Knowledge, attitude, and uptake related to human papillomavirus vaccination among young women in Germany recruited via a social media site.

    Science.gov (United States)

    Remschmidt, Cornelius; Walter, Dietmar; Schmich, Patrick; Wetzstein, Matthias; Deleré, Yvonne; Wichmann, Ole

    2014-01-01

    Many industrialized countries have introduced human papillomavirus (HPV) vaccination of young women, but vaccine uptake often remains suboptimal. This study aimed to investigate whether a social media site like Facebook is an appropriate tool to assess knowledge, attitude and uptake related to HPV vaccination in young women in Germany. Between December 2012 and January 2013 two different targeting strategies were implemented on Facebook, providing a link to an online questionnaire. Advertisements were displayed to female Facebook users aged 18-25 years living in Germany. During the simple targeting strategy, advertisements comprised health-related images along with various short titles and text messages. During the focused strategy, advertisements were targeted to users who in addition had certain fashion brands or pop stars listed on their profiles. The targeting strategies were compared with respect to participant characteristics. Univariate and multivariate analyses were used to identify factors associated with HPV vaccine uptake. A total of 1161 women participated. The two targeting strategies resulted in significant differences regarding educational status and migrant background. Overall, awareness of HPV was high, but only 53% received at least one vaccine dose. In multivariate analysis, HPV vaccine uptake was independently associated with a physician's recommendation and trust in vaccine effectiveness. Concerns of adverse effects were negatively associated with vaccine uptake. Social network recruitment permits fast and convenient access to young people. Sample characteristics can be manipulated by adjusting targeting strategies. There is further need for promoting knowledge of HPV vaccination among young women. Physicians have a major role in the vaccination decision-making process of young women.

  2. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  3. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

    DEFF Research Database (Denmark)

    Hägglund, Per; Matthiesen, R.; Elortza, F.

    2007-01-01

    and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

  4. Mapping the Binding Site for Escitalopram and Paroxetine in the Human Serotonin Transporter Using Genetically Encoded Photo-Cross-Linkers

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Bang-Andersen, Benny

    2017-01-01

    amber codon suppression in hSERT to encode the photo-cross-linking unnatural amino acid p-azido-l-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced cross-linking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective...... serotonin reuptake inhibitors (SSRIs). We find that the two antidepressant drugs exclusively cross-link to azF incorporated at the high-affinity binding site of hSERT, while cross-linking is not observed at the low-affinity binding site. Combined with previous homology models and recent structural data on h...

  5. Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

    Science.gov (United States)

    Pepper, Andrew R; Bruni, Antonio; Pawlick, Rena; Wink, John; Rafiei, Yasmin; Gala-Lopez, Boris; Bral, Mariusz; Abualhassan, Nasser; Kin, Tatsuya; Shapiro, A M James

    2017-10-01

    Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P islet function when transplanted under the KC (P islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

  6. In silico assessment of phosphorylation and O-β-GlcNAcylation sites in human NPC1 protein critical for Ebola virus entry.

    Science.gov (United States)

    Basharat, Zarrin; Yasmin, Azra

    2015-08-01

    Ebola is a highly pathogenic enveloped virus responsible for deadly outbreaks of severe hemorrhagic fever. It enters human cells by binding a multifunctional cholesterol transporter Niemann-Pick C1 (NPC1) protein. Post translational modification (PTM) information for NPC1 is crucial to understand Ebola virus (EBOV) entry and action due to changes in phosphorylation or glycosylation at the binding site. It is difficult and costly to experimentally assess this type of interaction, so in silico strategy was employed. Identification of phosphorylation sites, including conserved residues that could be possible targets for 21 predicted kinases was followed by interplay study between phosphorylation and O-β-GlcNAc modification of NPC1. Results revealed that only 4 out of 48 predicted phosphosites exhibited O-β-GlcNAc activity. Predicted outcomes were integrated with residue conservation and 3D structural information. Three Yin Yang sites were located in the α-helix regions and were conserved in studied vertebrate and mammalian species. Only one modification site S425 was found in β-turn region located near the N-terminus of NPC1 and was found to differ in pig, mouse, cobra and humans. The predictions suggest that Yin Yang sites may not be important for virus attachment to NPC1, whereas phosphosite 473 may be important for binding and hence entry of Ebola virus. This information could be useful in addressing further experimental studies and therapeutic strategies targeting PTM events in EBOV entry. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Strategy utilized for assessing baseline risks to human health from K-65 and metal oxide residues stored at the Fernald Site

    International Nuclear Information System (INIS)

    Harmon, J.E.; Janke, R.C.

    1995-01-01

    The U.S. Department of Energy (DOE) is responsible for cleanup activities at the Fernald Environmental Management Project (FEMP) site in southwestern Ohio. The 425-hectare site consists of a former 55-hectare Production Area, an adjacent Waste Storage Area and various support facilities. From 1952 until 1989, the FEMP processed uranium into metallic open-quotes feedclose quotes materials for other DOE facilities in the nation's defense program. In accordance with the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), the FEMP site is currently listed on the National Priorities List (NPL). To facilitate an expeditious cleanup effort, environmental issues associated with site cleanup are being managed under five operable units. This paper summarizes the risk assessment strategy employed to determine baseline human health risks associated with K-65 and metal oxide residues currently stored in Operable Unit 4. The K-65 and metal oxide residues were generated during the 1950s as a result of the extraction of uranium from uranium-bearing ores and concentrates. These residues are currently stored within Operable Unit 4 in concrete silos. Silos I and 2 contain approximately 6,120 cubic meters [m 3 ] (8,005 cubic yards [yd 3 ]) of K-65 residues, while silos 3 contains approximately 3890 m 3 (5,080 yd 3 ) of cold metal oxides. These concrete silos are beyond their design life and require remedial action. The risk assessment conducted for Operable Unit 4 constitutes the first detailed human health risk assessment to be approved by the Environmental Protection Agency (EPA) for the CERCLA clean-up effort at the FEMP Site. This paper discusses the FEMP's use of a Risk Information Quality Objective process in concert with the traditional risk assessment approach to determine baseline risk to human health and the environment posed by Operable Unit 4. A summary of the baseline risks to human health is also presented

  8. Strategy utilized for assessing baseline risks to human health from K-65 and metal oxide residues stored at the Fernald Site

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, J.E. [FERMCO, Cincinnati, OH (United States). Fernald Environmental Management Project; Janke, R.C.

    1995-04-01

    The U.S. Department of Energy (DOE) is responsible for cleanup activities at the Fernald Environmental Management Project (FEMP) site in southwestern Ohio. The 425-hectare site consists of a former 55-hectare Production Area, an adjacent Waste Storage Area and various support facilities. From 1952 until 1989, the FEMP processed uranium into metallic {open_quotes}feed{close_quotes} materials for other DOE facilities in the nation`s defense program. In accordance with the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), the FEMP site is currently listed on the National Priorities List (NPL). To facilitate an expeditious cleanup effort, environmental issues associated with site cleanup are being managed under five operable units. This paper summarizes the risk assessment strategy employed to determine baseline human health risks associated with K-65 and metal oxide residues currently stored in Operable Unit 4. The K-65 and metal oxide residues were generated during the 1950s as a result of the extraction of uranium from uranium-bearing ores and concentrates. These residues are currently stored within Operable Unit 4 in concrete silos. Silos I and 2 contain approximately 6,120 cubic meters [m{sup 3}] (8,005 cubic yards [yd{sup 3}]) of K-65 residues, while silos 3 contains approximately 3890 m{sup 3} (5,080 yd{sup 3}) of cold metal oxides. These concrete silos are beyond their design life and require remedial action. The risk assessment conducted for Operable Unit 4 constitutes the first detailed human health risk assessment to be approved by the Environmental Protection Agency (EPA) for the CERCLA clean-up effort at the FEMP Site. This paper discusses the FEMP`s use of a Risk Information Quality Objective process in concert with the traditional risk assessment approach to determine baseline risk to human health and the environment posed by Operable Unit 4. A summary of the baseline risks to human health is also presented.

  9. Structure of human milk bile salt activated lipase

    International Nuclear Information System (INIS)

    Baba, T.; Downs, D.; Jackson, K.W.; Tang, J.; Wang, Chi-Sun

    1991-01-01

    The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in λgt11 and λgt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDAN as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. The authors have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL

  10. The use of spectral skin reflectivity and laser doppler vibrometry data to determine the optimal site and wavelength to collect human vital sign signatures

    Science.gov (United States)

    Byrd, Kenneth A.; Kaur, Balvinder; Hodgkin, Van A.

    2012-06-01

    The carotid artery has been used extensively by researchers to demonstrate that Laser Doppler Vibrometry (LDV) is capable of exploiting vital sign signatures from cooperative human subjects at stando. Research indicates that, the carotid, although good for cooperative and non-traumatic scenarios, is one of the first vital signs to become absent or irregular when a casualty is hemorrhaging and in progress to circulatory (hypovolemic) shock. In an effort to determine the optimal site and wavelength to measure vital signs off human skin, a human subject data collection was executed whereby 14 subjects had their spectral skin reflectivity and vital signs measured at five collection sites (carotid artery, chest, back, right wrist and left wrist). In this paper, we present our findings on using LDV and re ectivity data to determine the optimal collection site and wavelength that should be used to sense pulse signals from quiet and relatively motionless human subjects at stando. In particular, we correlate maximum levels of re ectivity across the ensemble of 14 subjects with vital sign measurements made with an LDV at two ranges, for two scenarios.

  11. The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor

    OpenAIRE

    1990-01-01

    T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP ...

  12. Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Keishi Yamasaki

    Full Text Available A wide variety of drugs bind to human serum albumin (HSA at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand and ibuprofen (site II ligand at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a 'competitive-like' allosteric interaction in the case of the defatted HSA conformer to a 'nearly independent' binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a 'competitive-like' allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to 'nearly independent' binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

  13. The prevalence and concordance of human papillomavirus infection in different anogenital sites among men and women in Liuzhou, China: A population-based study.

    Science.gov (United States)

    Wei, Feixue; Li, Mingqiang; Wu, Xin; Yin, Kai; Lan, Jian; Sheng, Wei; Guo, Meng; Huang, Shoujie; Wang, Ying; Li, Yanping; Li, Rongcheng; Su, Yingying; Wu, Ting; Zhang, Jun; Xia, Ningshao

    2018-03-15

    Human papillomavirus (HPV) infection is the pathogenesis of anogenital cancers and genital warts in both men and women, whereas there is a scarcity of large studies focused on HPV prevalence in different anogenital sites of both sexes in the same population. From May to July 2014, 2,309 men and 2,378 women aged 18-55 were enrolled from communities in Liuzhou, China. Penis/glans penis/coronary sulcus (PGC) and perianal/anal canal (PA) specimens of men, and vaginal (VA), vulvar (VU) and PA specimens of women, were collected and genotyped for HPV. The prevalence of any HPV tested in PGC and PA samples from men and VA, VU and PA samples from women was 10.8%, 3.8%, 14.2%, 13.3% and 8.4%, respectively. The concordance of VA and VU was highest (kappa = 0.74), followed by VU and PA (0.44), VA and PA (0.38) and PGC and PA (0.14). Besides sex behavior, ever having used a towel supplied by a hotel was a risk factor for both external genital and PA HPV infection. Our data indicated that women were more of a major reservoir for oncogenic HPV infection of both genital sites and PA sites than was men. In both sexes, the genital sites were more likely than PA sites to harbor HPV infection. The concordance rates of HPV infection between genital sites and PA infection were poor. © 2017 UICC.

  14. Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites

    Directory of Open Access Journals (Sweden)

    Pierce Levi CT

    2009-01-01

    Full Text Available Abstract Background Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations. Results Using up-to-date databases containing all cancer-specific recurrent translocations, we have examined 444 unique pairs of genes involved in these translocations to determine the correlation of translocation breakpoints and fragile sites in the gene pairs. We found that over half (52% of translocation breakpoints in at least one gene of these gene pairs are mapped to fragile sites. Among these, we examined the DNA sequences within and flanking three randomly selected pairs of translocation-prone genes, and found that they exhibit characteristic features of fragile DNA, with frequent AT-rich flexibility islands and the potential of forming highly stable secondary structures. Conclusion Our study is the first to examine gene pairs involved in all recurrent chromosomal translocations observed in tumor cells, and to correlate the location of more than half of breakpoints to positions of known fragile sites. These results provide strong evidence to support a causative role for fragile sites in the generation of cancer-specific chromosomal rearrangements.

  15. Palaeo-ethnology, palaeo-environment and palaeo-climatology of Piaui, Brazil: palynological studies on human coprolites collected at the ''Pedra Furada'' site

    International Nuclear Information System (INIS)

    Chaves, S.; Renault-Miskovsky, J.

    1996-01-01

    Fossil human faeces were collected from the rock-shelter of ''Pedra Furada'' (Piaui, Brazil), which is considered nowadays as one of the most ancient prehistoric sites in America. These dated coprolites range from 8,500 to 7,000 BP and are evidence of an occupation phase connected with ''Serra Talhada'' cultural traditions I and II. These coprolites were analysed by palynological studies. The results have given palaeo-climatological and palaeo-environmental data with an emphasis on palaeo-ethnological aspects. Evidence emerges regarding the range of medicinal and food plants of the prehistoric humans who inhabited the site for some 1,500 years. (authors). 32 refs., 3 figs

  16. Identification and characterization of a cluster of transcription start sites located in the E6 ORF of human papillomavirus type 16

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Hansen, Christina N

    2003-01-01

    Human papillomavirus type 16 (HPV-16) is the prototype strain among the malignant types of HPV in the western world. The main promoter, P97, located in front of the E6 ORF, has been shown to control expression of the oncogenes E6 and E7. These oncogenes are expressed continuously in HPV-16......-transformed cells. In contrast to malignant HPV types, non-malignant HPV types have separate promoters driving the expression of E6 and E7. Experiments have shown that the translation of E7 is more efficient from monocistronic than bicistronic transcripts encoding both E6 and E7. Here, identification...... of a cluster of transcription start sites located in the E6 ORF of HPV-16 is presented. Transcripts from this region contain the E7 ORF as the first reading frame. The cluster consists of multiple transcription start sites located around nt 441. Additional transcription start sites were identified in a cluster...

  17. Climate, Environment and Early Human Innovation: Stable Isotope and Faunal Proxy Evidence from Archaeological Sites (98-59ka in the Southern Cape, South Africa.

    Directory of Open Access Journals (Sweden)

    Patrick Roberts

    Full Text Available The Middle Stone Age (MSA of southern Africa, and in particular its Still Bay and Howiesons Poort lithic traditions, represents a period of dramatic subsistence, cultural, and technological innovation by our species, Homo sapiens. Climate change has frequently been postulated as a primary driver of the appearance of these innovative behaviours, with researchers invoking either climate instability as a reason for the development of buffering mechanisms, or environmentally stable refugia as providing a stable setting for experimentation. Testing these alternative models has proved intractable, however, as existing regional palaeoclimatic and palaeoenvironmental records remain spatially, stratigraphically, and chronologically disconnected from the archaeological record. Here we report high-resolution records of environmental shifts based on stable carbon and oxygen isotopes in ostrich eggshell (OES fragments, faunal remains, and shellfish assemblages excavated from two key MSA archaeological sequences, Blombos Cave and Klipdrift Shelter. We compare these records with archaeological material remains in the same strata. The results from both sites, spanning the periods 98-73 ka and 72-59 ka, respectively, show significant changes in vegetation, aridity, rainfall seasonality, and sea temperature in the vicinity of the sites during periods of human occupation. While these changes clearly influenced human subsistence strategies, we find that the remarkable cultural and technological innovations seen in the sites cannot be linked directly to climate shifts. Our results demonstrate the need for scale-appropriate, on-site testing of behavioural-environmental links, rather than broader, regional comparisons.

  18. Computational Characterization of Small Molecules Binding to the Human XPF Active Site and Virtual Screening to Identify Potential New DNA Repair Inhibitors Targeting the ERCC1-XPF Endonuclease

    Directory of Open Access Journals (Sweden)

    Francesco Gentile

    2018-04-01

    Full Text Available The DNA excision repair protein ERCC-1-DNA repair endonuclease XPF (ERCC1-XPF is a heterodimeric endonuclease essential for the nucleotide excision repair (NER DNA repair pathway. Although its activity is required to maintain genome integrity in healthy cells, ERCC1-XPF can counteract the effect of DNA-damaging therapies such as platinum-based chemotherapy in cancer cells. Therefore, a promising approach to enhance the effect of these therapies is to combine their use with small molecules, which can inhibit the repair mechanisms in cancer cells. Currently, there are no structures available for the catalytic site of the human ERCC1-XPF, which performs the metal-mediated cleavage of a DNA damaged strand at 5′. We adopted a homology modeling strategy to build a structural model of the human XPF nuclease domain which contained the active site and to extract dominant conformations of the domain using molecular dynamics simulations followed by clustering of the trajectory. We investigated the binding modes of known small molecule inhibitors targeting the active site to build a pharmacophore model. We then performed a virtual screening of the ZINC Is Not Commercial 15 (ZINC15 database to identify new ERCC1-XPF endonuclease inhibitors. Our work provides structural insights regarding the binding mode of small molecules targeting the ERCC1-XPF active site that can be used to rationally optimize such compounds. We also propose a set of new potential DNA repair inhibitors to be considered for combination cancer therapy strategies.

  19. Climate, Environment and Early Human Innovation: Stable Isotope and Faunal Proxy Evidence from Archaeological Sites (98-59ka) in the Southern Cape, South Africa.

    Science.gov (United States)

    Roberts, Patrick; Henshilwood, Christopher S; van Niekerk, Karen L; Keene, Petro; Gledhill, Andrew; Reynard, Jerome; Badenhorst, Shaw; Lee-Thorp, Julia

    2016-01-01

    The Middle Stone Age (MSA) of southern Africa, and in particular its Still Bay and Howiesons Poort lithic traditions, represents a period of dramatic subsistence, cultural, and technological innovation by our species, Homo sapiens. Climate change has frequently been postulated as a primary driver of the appearance of these innovative behaviours, with researchers invoking either climate instability as a reason for the development of buffering mechanisms, or environmentally stable refugia as providing a stable setting for experimentation. Testing these alternative models has proved intractable, however, as existing regional palaeoclimatic and palaeoenvironmental records remain spatially, stratigraphically, and chronologically disconnected from the archaeological record. Here we report high-resolution records of environmental shifts based on stable carbon and oxygen isotopes in ostrich eggshell (OES) fragments, faunal remains, and shellfish assemblages excavated from two key MSA archaeological sequences, Blombos Cave and Klipdrift Shelter. We compare these records with archaeological material remains in the same strata. The results from both sites, spanning the periods 98-73 ka and 72-59 ka, respectively, show significant changes in vegetation, aridity, rainfall seasonality, and sea temperature in the vicinity of the sites during periods of human occupation. While these changes clearly influenced human subsistence strategies, we find that the remarkable cultural and technological innovations seen in the sites cannot be linked directly to climate shifts. Our results demonstrate the need for scale-appropriate, on-site testing of behavioural-environmental links, rather than broader, regional comparisons.

  20. Fluorescence spectrometric studies on the binding of puerarin to human serum albumin using warfarin, ibuprofen and digitoxin as site markers with the aid of chemometrics

    International Nuclear Information System (INIS)

    Zhang Guowen; Zhao Nan; Wang Lin

    2011-01-01

    The interaction of puerarin with human serum albumin (HSA) in pH 7.4 Tris-HCl buffer has been investigated by fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. The results revealed the presence of static type of quenching mechanism in the binding of puerarin to HSA. The association constants (K a ) between puerarin and HSA were obtained according to Modified Stern-Volmer equation. The calculated thermodynamic parameters indicated that the binding of puerarin to HSA was driven mainly by hydrophobic interaction. The competitive experiments of site markers suggested that the binding site of puerarin to HSA was located in the region of subdomain IIA (sudlow site I). Further, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectra data of the competitive interaction between puerarin and warfarin with HSA. The concentration information for the three reaction components, warfarin, puerarin and puerarin-HSA, in the system at equilibrium was obtained simultaneously. The PARAFAC analysis indicated that puerarin in the puerarin-HSA complex was displaced by warfarin, which confirmed the binding site of puerarin to HSA was located in site I. Moreover, the results of CD and FT-IR spectra demonstrated that the secondary structure of HSA was changed in the presence of puerarin. - Highlights: → Puerarin can quench the fluorescence of human serum albumin (HSA). → The HSA fluorescence is quenched by puerarin through a static quenching mechanism. → The binding of puerarin to HSA is driven mainly by hydrophobic interaction. → The parallel factor analysis confirms that puerarin is located in site I of HSA. → The binding of puerarin to HSA induces changes in the secondary structure of HSA.

  1. Environmental impact and site-specific human health risks of chromium in the vicinity of a ferro-alloy manufactory, China.

    Science.gov (United States)

    Wang, Zhen-xing; Chen, Jian-qun; Chai, Li-yuan; Yang, Zhi-hui; Huang, Shun-hong; Zheng, Yu

    2011-06-15

    Previous studies often neglected the direct exposure to soil heavy metals in human health risk assessment. The purpose of this study was to assess the environmental impact and site-specific health risks of chromium (Cr) by both direct and indirect exposure assessment method. Results suggested that total Cr was shown a substantial buildup with a significant increase in the industrial and cultivated soils (averaged 1910 and 986 mg kg(-1), respectively). The Cr contents of vegetables exceeded the maximum permissible concentration by more than four times in every case. Human exposure to Cr was mainly due to dietary food intake in farming locations and due to soil ingestion in both industrial and residential sites. Soil ingestion was the main contributor pathway for direct exposure, followed by inhalation, and then dermal contact. The highest risks of vegetable ingestion were associated with consumption of Chinese cabbage. The results also indicated that plant tissues are able to convert the potentially toxic Cr (VI) species into the non-toxic Cr (III) species. The analyses of human health risks indicated that an important portion of the population is at risk, especially in the industrial site. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome

    International Nuclear Information System (INIS)

    Economou, E.P.; Bergen, A.W.; Warren, A.C.; Antonarakis, S.E.

    1990-01-01

    To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, the authors hypothesize that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. Analysis of the 3' ends of three specific Alu sequences showed two occurrences, one in the adenosine deaminase gene and other in the β-globin pseudogene, were polymorphic. This novel class of polymorphism, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome

  3. An in vivo approach for globally estimating the drug flow between blood and tissue for nafamostat mesilate: the main hydrolysis site determination in human.

    Science.gov (United States)

    Cao, Yan-Guang; Chen, Yuan-Cheng; Hao, Kun; Zhang, Ming; Liu, Xiao-Quan

    2008-11-01

    Nafamostat mesilate, an ester drug with extensive hydrolysis in vivo, exhibits species difference in the relative contribution for its hydrolysis in blood and tissues. For the rat, the main hydrolysis site may be blood and human may be tissue (mainly by liver). The paper gave in vivo evidence that human tissue may give more contribution for its hydrolysis. In the initial phase of drug administration, the drug accumulating level in tissue was low; the efflux fraction from tissue into blood can be ignorable comparing with the drug influx into tissue. Based on urine and plasma metabolite analysis, we concluded that in the initial phase almost all the drug hydrolysis in blood was excreted into urine. Then according to the initial urine metabolite analysis, we can estimate the drug hydrolysis rate in blood. The rate of drug diffusion from blood into tissues can be deduced based on the mass balance analysis of the initial blood drug. With the estimated rate constants, the drug efflux from tissues into blood was calculated according to equation: OFT-B (efflux from tissues) = OFB-U (blood hydrolysis fraction)+OFB-T (influx into tissues)-DB (hydrolysis in blood). The net flow (influent flux minus effluent flux) represented the drug hydrolysis fraction in tissue. As the result indicated, in human about 20% drug administrated was hydrolyzed in blood and nearly 80% in tissues. The relative hydrolysis fraction indicated that the main hydrolysis site in human body may locate in tissue, which was different to rats.

  4. Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

    Science.gov (United States)

    Asakura, H; Makino, S; Kobori, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K

    2001-08-01

    Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

  5. Concentration and transportation of heavy metals in vegetables and risk assessment of human exposure to bioaccessible heavy metals in soil near a waste-incinerator site, South China.

    Science.gov (United States)

    Li, Ning; Kang, Yuan; Pan, Weijian; Zeng, Lixuan; Zhang, Qiuyun; Luo, Jiwen

    2015-07-15

    There is limited study focusing on the bioaccumulation of heavy metals in vegetables and human exposure to bioaccessible heavy metals in soil. In the present study, heavy metal concentrations (Cr, Ni, Cu, Pb and Cd) were measured in five types of vegetables, soil, root, and settled air particle samples from two sites (at a domestic waste incinerator and at 20km away from the incinerator) in Guangzhou, South China. Heavy metal concentrations in soil were greater than those in aerial parts of vegetables and roots, which indicated that vegetables bioaccumulated low amount of heavy metals from soil. The similar pattern of heavy metal (Cr, Cd) was found in the settled air particle samples and aerial parts of vegetables from two sites, which may suggest that foliar uptake may be an important pathway of heavy metal from the environment to vegetables. The highest levels of heavy metals were found in leaf lettuce (125.52μg/g, dry weight) and bitter lettuce (71.2μg/g) for sites A and B, respectively, followed by bitter lettuce and leaf lettuce for sites A and B, respectively. Swamp morning glory accumulated the lowest amount of heavy metals (81.02μg/g for site A and 53.2μg/g for site B) at both sites. The bioaccessibility of heavy metals in soil ranged from Cr (2%) to Cu (71.78%). Risk assessment showed that Cd and Pb in soil samples resulted in the highest non-cancer risk and Cd would result in unacceptable cancer risk for children and risk. The non-dietary intake of soil was the most important exposure pathway, when the bioaccessibility of heavy metals was taken into account. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

  7. Tracking Human-Induced Landscape Disturbance at the Nasca Lines UNESCO World Heritage Site in Peru with COSMO-SkyMed InSAR

    Directory of Open Access Journals (Sweden)

    Francesca Cigna

    2018-04-01

    Full Text Available The “Lines and Geoglyphs of Nasca and Palpa” in Peru are among the most well-known UNESCO World Heritage Sites globally, and an exemplar of site where heritage assets cannot be separated from their natural and anthropogenic environment. The site is exposed to interactions with natural processes, as well as human presence. In this work, 3-m resolution synthetic aperture radar (SAR StripMap HIMAGE HH-polarised scenes acquired by the X-band COSMO-SkyMed constellation are exploited to track two events of human-induced landscape disturbance that occurred in December 2014 and January 2018. Pre-, cross-, and post-event interferometric SAR (InSAR pairs characterised by small temporal and normal baselines allow the detection of temporal decorrelation associated with the two events, the extent and time reference of which match with online photographic and video evidence, published literature, web news, and press releases by the Ministry of Culture in Peru. Further elements enhancing the understanding of the 2018 event come from 10-m resolution Sentinel-2B satellite data that reveal the occurrence of apparent changes of surface reflectance due to uncovering of the light grey-yellow clay underneath the darker pebble constituting the fragile surface of the Pampa de Jumana. This scientific study confirms that SAR imagery archives, such as those being built by COSMO-SkyMed for Nasca, prove valuable for the retrospective analysis and digital recording of human-induced landscape disturbance events from space. These archives therefore act as essential sources of geospatial information on the conservation history of heritage sites and assets.

  8. Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

    Directory of Open Access Journals (Sweden)

    Nicolas Grandchamp

    Full Text Available Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.

  9. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    Directory of Open Access Journals (Sweden)

    Gowda Veerabasappa T

    2007-12-01

    Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  10. Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules.

    Science.gov (United States)

    Nurden, A T; Horisberger, M; Savariau, E; Caen, J P

    1980-10-15

    Washed human platelets have been incubated with the lectins WGA, ConA and RCA1, adsorbed to different-sized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.

  11. Distinct molecular sites of anaesthetic action: pentobarbital block of human brain sodium channels is alleviated by removal of fast inactivation

    NARCIS (Netherlands)

    Wartenberg, H. C.; Urban, B. W.; Duch, D. S.

    1999-01-01

    Fast inactivation of sodium channel function is modified by anaesthetics. Its quantitative contribution to the overall anaesthetic effect is assessed by removing the fast inactivation mechanism enzymatically. Sodium channels from human brain cortex were incorporated into planar lipid bilayers. After

  12. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen

    2016-01-01

    with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within...

  13. Docking and molecular dynamics studies of peripheral site ligand–oximes as reactivators of sarin-inhibited human acetylcholinesterase

    NARCIS (Netherlands)

    Almeida, J.S.F.D. de; Cuya Guizado, T.R.; Guimarães, A.P.; Ramalho, T.C.; Gonçalves, A.S.; Koning, M.C. de; França, T.C.C.

    2016-01-01

    In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor

  14. Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: A test case with some Sudlow's site I ligands

    International Nuclear Information System (INIS)

    Poór, Miklós; Li, Yin; Matisz, Gergely; Kiss, László; Kunsági-Máté, Sándor; Kőszegi, Tamás

    2014-01-01

    Albumin, the most abundant plasma protein is an approximately 67 kDa sized water-soluble macromolecule. Since several drugs and xenobiotics circulate in the blood at least partially in albumin-bound form, albumin plays a key role in the pharmacokinetics/toxicokinetics of these chemicals. Most of the drugs and xenobiotics are Sudlow's site I ligands. In numerous studies, bovine serum albumin (BSA) is used for modeling albumin–ligand interactions and the results are extrapolated to human serum albumin (HSA). Furthermore, only limited information is available related to albumin–ligand interactions of different albumin species. Therefore, in our study, we have focused on the quantification of differences between bovine, human and rat serum albumin (RSA) using four Sudlow's site I ligands (luteolin, ochratoxin A, phenylbutazone and warfarin). Interactions were analyzed by fluorescence spectroscopy. Stability constants as well as competing capacities of the ligands were determined, and thermodynamic study was also performed. Our results highlight that there could be major differences between BSA, HSA and RSA in their ligand binding properties. Based on our observations we emphasize that in molecular aspects BSA behaves considerably differently from HSA or from albumins of other species therefore, it is strongly recommended to apply at least some confirmatory measurements when data obtained from other species are attempted to be extrapolated to HSA. -- Highlights: • Albumin–ligand interactions of human, bovine and rat albumins were studied. • Four Sudlow's site I ligands were tested by fluorescence spectroscopy. • Substantial differences were found in stability constants among albumin complexes. • Competing capacity of ligands showed major differences in the studied species. • Data obtained for BSA cannot be directly extrapolated to human albumin

  15. Noctis Landing: A Proposed Landing Site/Exploration Zone for Human Missions to the Surface of Mars

    Science.gov (United States)

    Lee, Pascal; Acedillo, Shannen; Braham, Stephen; Brown, Adrian; Elphic, Richard; Fong, Terry; Glass, Brian; Hoftun, Christopher; Johansen, Brage W.; Lorber, Kira; hide

    2015-01-01

    The proposed Noctis Landing Site/Exploration Zone (LS/EZ) is shown in Figure 1. Our preliminary study suggests that the proposed site meets all key Science and Resources (incl. Civil Engineering) requirements. The site is of significant interest, as the EZ not only offers a large number and wide range of regions of interest (ROIs) for short-term exploration, it is also located strategically at the crossroads between Tharsis and Valles Marineris, which are key for long-term exploration. The proposed site contains Regions of Interest (ROIs) that meet the following Science requirements: -­- Access to (1) deposits with a high preservation potential for evidence of past habitability and fossil biosignatures and (2) sites that are promising for present habitability. The site presents a wide variety of ROIs qith likely aqueous features and deposits, including sinous channels and valleys, slope gullies, lobate debris aprons, impact craters with lobate ejecta flows, and "bathtub ring" deposits. Neutron spectrometry also suggests hydrogen is present within the topmost 0.3 m or so of 4 to 10 wt% WEH (Water Equivalent Hydrogen). -­- Noachian and/or Hesperian rocks in a stratigraphic context that have a high likelihood of containing trapped atmospheric gases. Collapsed canyon rim material with preserved stratigraphy is abundantly present and accessible. -­- Exposures of at least two crustal units that have regional or global extents, that are suitable for radiometric dating, and that have relative ages that sample a significant range of martian geological time. Canyons floors in Ius Chasma, Tithonium Chasma, and plateau tops on Tharsis and in Sinai Planum offer access to distinct crustal units of regional extent. -­- Access to outcrops with linked morphological and/or geochemical signatures indicative of aqueous or groundwater/ mineral interactions. Iron and sulfur-bearing deposits on canyon floors in Noctis Labyrinthus, and in Ius Chasma (IC) and Tithonium Chasma (TC

  16. A new luminescence dating chronology for the Rhafas cave site (NE Morocco): Insights into Palaeolithic human cultural change under varying palaeoenvironmental conditions in the Maghreb

    Science.gov (United States)

    Dörschner, Nina; Fitzsimmons, Kathryn E.; Ditchfield, Peter; McLaren, Sue J.; Steele, Teresa E.; Zielhofer, Christoph; McPherron, Shannon P.; Bouzouggar, Abdeljalil; Hublin, Jean-Jacques

    2016-04-01

    Archaeological sites in northern Africa provide a rich record that is of increasing importance for current debates relating to the origins of modern human behaviour and to Out of Africa human dispersal events. Particular interest is placed on the cultural transition between the North African Middle Stone Age (MSA) and Late Stone Age (LSA), and the need for accurately defined chronologies, however the timing and nature of Palaeolithic human behaviour and dispersal across north-western Africa (the Maghreb) and potential correlation with environmental conditions remain poorly understood. The inland cave site of Rhafas (Morocco) preserves a long stratified sequence providing valuable chronological information about cultural changes in the Maghreb spanning the North African MSA through to the Neolithic. In this study, we apply optically stimulated luminescence (OSL) dating on sand-sized quartz grains to the cave deposits of Rhafas as well as to a section on the terrace in front of the cave entrance. Single grain OSL dating reliably constrains the timing of technocomplexes beyond the limits of radiocarbon by directly dating sediment associated with archaeological traces. We combine OSL dating with multi-proxy geological investigations (XRF, grain size analyses, stable isotopes, thin sections) to investigate site formation processes and reconstruct palaeoenvironmental conditions during human occupation phases at Rhafas. Our results indicate that the occupation of the site started at least in MIS 6 during a phase of relatively arid environmental conditions. Climatic amelioration after c.140 ka is associated with a change in sediment geochemistry at the site, most likely linked to a change in sediment source due to shifting wind directions. Tanged pieces - typical for the classical Aterian technocomplex - start to occur in the archaeological sequence in MIS 5, consistent with previously published chronological data from the Maghreb. From 55 ka, climatic conditions were

  17. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    International Nuclear Information System (INIS)

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-01-01

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners

  18. Human biological monitoring as demonstrated by means of a heavy-metal polluted abandoned site; Human-Biomonitoring am Beispiel einer Schwermetallaltlast

    Energy Technology Data Exchange (ETDEWEB)

    Elison, M; Schulte-Hostede, S [GSF-Forschungzentrum fuer Umwelt und Gesundheit Neuherberg (Germany). Inst. fuer Oekologische Chemie

    1998-12-31

    Models for estimating exposure permit to make a rough assessment of the risk emanating from a contaminated area. But it must not be overlooked that such models are fraught with considerable weaknesses.- In studies such as the one described, concerned citizens should additionally be examined in order to obtain supplementary information and to aid interpretation. Such human biological monitoring makes sense only if the persons in question actually live in the contaminated areas, so that a higher exposure may reasonably be expected. Human biological monitoring is to help assess the inner exposure of human beings to pollutants emanating from the contaminated area. (orig./SR) [Deutsch] Mit Hilfe von Modellen zur Expositionsabschaetzung ist man nach den oben dargestellten Vorgehensweisen in der Lage, eine orientierende Bewertung des von einer kontaminierten Flaeche ausgehenden Risikos vorzunehmen. Dabei ist jedoch zu beruecksichtigen, dass solche Modelle mit erheblichen Schwachstellen belastet sind. Zur Ergaenzung und Interpretationshilfe sind bei Untersuchungen wie der hier vorgestellten auch Untersuchungen an den betroffenen Buergen vorzunehmen. Dieses Human-Biomonitoring hat nur dort einen Sinn, wo sichergestellt ist, dass die Menschen dort tatsaechlich auf belasteten Flaechen leben und damit eine erhoehte Belastung der Menschen anzunehmen ist. Das Human-Biomonitoring soll eine Abschaetzung der inneren Belastung des Menschen mit Schadstoffen, die von der kontaminierten Flaeche herruehren, ermoeglichen. (orig./SR)

  19. Human biological monitoring as demonstrated by means of a heavy-metal polluted abandoned site; Human-Biomonitoring am Beispiel einer Schwermetallaltlast

    Energy Technology Data Exchange (ETDEWEB)

    Elison, M.; Schulte-Hostede, S. [GSF-Forschungzentrum fuer Umwelt und Gesundheit Neuherberg (Germany). Inst. fuer Oekologische Chemie

    1997-12-31

    Models for estimating exposure permit to make a rough assessment of the risk emanating from a contaminated area. But it must not be overlooked that such models are fraught with considerable weaknesses.- In studies such as the one described, concerned citizens should additionally be examined in order to obtain supplementary information and to aid interpretation. Such human biological monitoring makes sense only if the persons in question actually live in the contaminated areas, so that a higher exposure may reasonably be expected. Human biological monitoring is to help assess the inner exposure of human beings to pollutants emanating from the contaminated area. (orig./SR) [Deutsch] Mit Hilfe von Modellen zur Expositionsabschaetzung ist man nach den oben dargestellten Vorgehensweisen in der Lage, eine orientierende Bewertung des von einer kontaminierten Flaeche ausgehenden Risikos vorzunehmen. Dabei ist jedoch zu beruecksichtigen, dass solche Modelle mit erheblichen Schwachstellen belastet sind. Zur Ergaenzung und Interpretationshilfe sind bei Untersuchungen wie der hier vorgestellten auch Untersuchungen an den betroffenen Buergen vorzunehmen. Dieses Human-Biomonitoring hat nur dort einen Sinn, wo sichergestellt ist, dass die Menschen dort tatsaechlich auf belasteten Flaechen leben und damit eine erhoehte Belastung der Menschen anzunehmen ist. Das Human-Biomonitoring soll eine Abschaetzung der inneren Belastung des Menschen mit Schadstoffen, die von der kontaminierten Flaeche herruehren, ermoeglichen. (orig./SR)

  20. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

    International Nuclear Information System (INIS)

    Xiang, Shengyan; Liu, Zhaojun; Zhang, Baozhen; Zhou, Jing; Zhu, Bu-Dong; Ji, Jiafu; Deng, Dajun

    2010-01-01

    Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively

  1. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

    Science.gov (United States)

    Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

    2014-09-01

    Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

  2. Regulatory domain or CpG site variation in SLC12A5, encoding the chloride transporter KCC2, in human autism and schizophrenia

    Directory of Open Access Journals (Sweden)

    Nancy D Merner

    2015-10-01

    Full Text Available Many encoded gene products responsible for neurodevelopmental disorders (NDs like autism spectrum disorders (ASD, schizophrenia (SCZ, intellectual disability (ID, and idiopathic generalized epilepsy (IGE converge on networks controlling synaptic function. An increase in KCC2 (SLC12A5 Cl- transporter activity drives the developmental GABA excitatory-inhibitory sequence, but the role of KCC2 in human NDs is essentially unknown. Here, we report two rare, non-synonymous (NS, functionally-impairing variants in the KCC2 C-terminal regulatory domain (CTRD in human ASD (R952H and R1049C and SCZ (R952H previously linked with IGE and familial febrile seizures, and another novel NS KCC2 variant in ASD (R1048W with highly-predicted pathogenicity. Exome data from 2517 simplex families in the ASD Simon Simplex Collection revealed significantly more KCC2 CTRD variants in ASD cases than controls, and interestingly, these were more often synonymous and predicted to disrupt or introduce a CpG site. Furthermore, full gene analysis showed ASD cases are more likely to contain rare KCC2 variants affecting CpG sites than controls. These data suggest genetically-encoded dysregulation of KCC2-dependent GABA signaling may contribute to multiple human NDs.

  3. A dominant control region from the human β-globin locus conferring integration site-independent gene expression.

    OpenAIRE

    Talbot, D.; Collis, P.; Antoniou, Michael; Vidal, M.; Grosveld, Frank; Greaves, David

    1989-01-01

    textabstractThe regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell lines and transgenic animals has turned out to be at a low level relative to that of endogenous genes, and influenced by the chromosomal site of insertion of the exogenous construct. We have previo...

  4. The mastermind approach to CNS drug therapy: translational prediction of human brain distribution, target site kinetics, and therapeutic effects

    OpenAIRE

    de Lange, Elizabeth CM

    2013-01-01

    Despite enormous advances in CNS research, CNS disorders remain the world?s leading cause of disability. This accounts for more hospitalizations and prolonged care than almost all other diseases combined, and indicates a high unmet need for good CNS drugs and drug therapies. Following dosing, not only the chemical properties of the drug and blood?brain barrier (BBB) transport, but also many other processes will ultimately determine brain target site kinetics and consequently the CNS effects. ...

  5. Differential labeling of dopamine and sigma sites by [3H]nemonapride and [3H]raclopride in postmortem human brains.

    Science.gov (United States)

    Tang, S W; Helmeste, D M; Fang, H; Li, M; Vu, R; Bunney, W; Potkin, S; Jones, E G

    1997-08-08

    The difference between the binding of [3H]nemonapride and [3H]raclopride has been used to quantify dopamine D4 receptors in postmortem schizophrenic brain studies. Recent work, however, has suggested that at least part of the differential between [3H]nemonapride and [3H]raclopride binding may represent sigma rather than D4 receptor sites. We applied the nemonapride-raclopride subtraction method to postmortem, non-schizophrenic human striatum to examine the variation in dopaminergic receptor binding labeled by these ligands. Variation in sigma receptor binding labeled by [3H]nemonapride was studied in frontal cortex, striatum and cerebellum. Specific binding was defined by sulpiride (dopamine receptor ligand), PPAP (sigma receptor ligand) and haloperidol (mixed dopaminergic/sigma agent), respectively. Haloperidol defined a combination of sites, which were approximately the sum of the dopaminergic and sigma components defined by sulpiride and PPAP, respectively. Significant inter-individual variation in the amount of specific binding for dopaminergic and sigma receptor sites was observed. However, no significant nor consistent observation of striatal dopamine D4 receptors or D4-like binding sites was observed in the striatum even though two independent sets of tissues, with different dissections were used. The inconsistencies in some previous postmortem studies appear to be at least partially explained by the inclusion of both sigma and dopaminergic components in [3H]nemonapride binding and the inherent high inter-individual variability of the different components.

  6. Tyrosine411 and Arginine410 of Human Serum Albumin Play an Important Role in the Binding of Sodium 4-Phenylbutyrate to Site II.

    Science.gov (United States)

    Enokida, Taisuke; Yamasaki, Keishi; Okamoto, Yuko; Taguchi, Kazuaki; Ishiguro, Takako; Maruyama, Toru; Seo, Hakaru; Otagiri, Masaki

    2016-06-01

    Sodium 4-phenylbutyrate (PB) has many pharmacological activities; therefore extending its clinical use to the treatment of a wider variety of diseases would be desirable. However, our knowledge of the binding of PB to plasma proteins is not extensive. To address this issue in more detail, we characterized the protein binding of PB. Binding experiments showed that PB mainly binds to human serum albumin (HSA) in plasma. PB was also found to bind to a single site on HSA, which was identified as site II by fluorescent probe displacement experiment. Furthermore, an appropriate alkyl chain length and a carboxylic group in the PB structure were required for PB binding to HSA, suggesting that hydrophobic (and van der Waals) and electrostatic interactions are involved as binding modes. The contributions of hydrogen bonding and/or van der Waals interactions were also indicated by thermodynamic analyses. Tyrosine411 and arginine410 were identified as being involved in the binding of PB to site II, based on binding experiments using chemically modified- and mutant-HSA preparations. In conclusion, the available evidence indicates that PB binds to site II of HSA with assistance by multiple forces and that tyrosine411 and arginine410 both play important roles in this phenomenon. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  7. Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

    DEFF Research Database (Denmark)

    Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

    2000-01-01

    Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

  8. Measurement of enzyme-sensitive sites in uv- or. gamma. -irradiated human cells using Micrococcus luteus extracts

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, M C; Smith, B P; Smith, P J

    1979-01-01

    The study of DNA damage and its enzymatic repair has undergone rapid expansion in recent years. Laboratory observations at the molecular level have been facilitated greatly by the availability of a battery of physicochemical techniques capable of monitoring hallmarks of different repair mechanisms. One technique exploits the unique ability of certain putative repair enzymes (endonucleases and DNA glycosylases of prokaryotic origin) to selectively attack DNA at sites containing altered base or sugar residues; the sites are subsequently observed as single-strand break, by velocity sedimentatn of the DNA in an alkaline sucrose gradient. Incubation of carcinogen-treated cell cultures for varying times, followed by enzymatic analysis of their radionuclide-labeled DNA, yields the time course of disappearace of such sites; this is taken as an indirect expression of the kinetics of lesion repair. Although there are several variations of the enzymatic assay two basic protocols are in current use. The only major difference is the way in which the damaged DNA is treated with the lesion-detecting enzyme(s). In one protocol this is achieved by rendering the cells porous to extracellular proteins prior to incubation with the test enzyme(s). In the second protocol the damaged DNA is extracted from the cells and is then exposed to the lesion-recognizing enzyme(s) in vitro. The enzymatic assay developed in our laboratory follows this second protocol, and the procedure is described.

  9. Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT).

    Science.gov (United States)

    Walker, D K; Bowers, S J; Mitchell, R J; Potchoiba, M J; Schroeder, C M; Small, H F

    2008-10-01

    1. Growing knowledge of the pathogenesis of human immunodeficiency virus (HIV)-1 infection has led to the identification of potential virus sanctuary sites within the central nervous system and gut-associated lymphoid tissue. 2. Maraviroc is a novel CCR5 antagonist for the treatment of HIV-1 infection. Disposition studies have been performed within the preclinical testing of maraviroc to determine its distribution to these anatomical sites. 3. Maraviroc, which is a substrate of the efflux transporter P-glycoprotein, shows limited distribution to the central nervous system as evidenced by cerebrospinal fluid concentrations that were 10% of the free plasma concentration following intravenous infusion to rats. Tissue distribution studies also indicated limited distribution of radioactivity into brain tissue of rats. 4. Radioactivity in gut-associated lymphoid tissue lymph nodes exceeded the concentrations in blood and concentrations in the contents of thoracic ducts of the lymphatic system were similar to blood levels following intravenous administration to rats.

  10. Utilizing Immersive Visualization Systems: How to Dynamically Revolutionize Site-based Professional Development Experiences within Human Resources Management?

    Science.gov (United States)

    Craft, Kirby A.

    2009-01-01

    How can we train today's workforce with innovative technologies when families are surrounded by state-of-the-art video games and high-definition televisions? Human resource managers and administrators are faced with difficult challenges to prepare beneficial and relevant professional development exercises that engage the minds of their employees.…

  11. Teratoma Formation by Human Embryonic Stem Cells is site-dependent and enhanced by the presence of Matrigel

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Harkness, Linda M; Frandsen, Ulrik

    2008-01-01

    When implanted into immunodeficient mice, human embryonic stem cells (hESC) give rise to teratoma, tumour-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data...

  12. Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities

    Czech Academy of Sciences Publication Activity Database

    Ambro, L.; Pevala, V.; Ondrovičová, G.; Bellová, J.; Kunová, N.; Kutejová, Eva; Bauer, J.

    2014-01-01

    Roč. 281, č. 7 (2014), s. 1784-1797 ISSN 1742-464X Institutional support: RVO:61388971 Keywords : ATP-dependent protease * glycine loop * human Lon protease Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  13. The Hominin Sites and Paleolakes Drilling Project : inferring the environmental context of human evolution from eastern African rift lake deposits

    NARCIS (Netherlands)

    Cohen, A.; Campisano, C.; Arrowsmith, R.; Asrat, A.; Behrensmeyer, A. K.; Deino, A.; Feibel, C.; Hill, A.; Johnson, R.; Kingston, J.; Lamb, H.; Lowenstein, T.; Noren, A.; Olago, D.; Owen, R. B.; Potts, R.; Reed, K.; Renaut, R.; Schäbitz, F.; Tiercelin, J.-J.; Trauth, M. H.; Wynn, J.; Ivory, S.; Brady, K.; O'Grady, R.; Rodysill, J.; Githiri, J.; Russell, J.; Foerster, V.; Dommain, R.; Rucina, S.; Deocampo, D.; Russell, J.; Billingsley, A.; Beck, C.; Dorenbeck, G.; Dullo, L.; Feary, D.; Garello, D.; Gromig, R.; Johnson, T.; Junginger, A.; Karanja, M.; Kimburi, E.; Mbuthia, A.; Mccartney, T.; Mcnulty, E.; Muiruri, V.; Nambiro, E.; Negash, E. W.; Njagi, D.; Wilson, J. N.; Rabideaux, N.; Raub, T.; Sier, M. J.; Smith, P.; Urban, J.; Warren, M.; Yadeta, M.; Yost, C.; Zinaye, B.

    2016-01-01

    The role that climate and environmental history may have played in influencing human evolution has been the focus of considerable interest and controversy among paleoanthropologists for decades. Prior attempts to understand the environmental history side of this equation have centered around the

  14. Location and characterization of the warfarin binding site of human serum albumin A comparative study of two large fragments

    NARCIS (Netherlands)

    Bos, O.J.M.; Remijn, J.P.M.; Fischer, M.J.E.; Wilting, J.; Janssen, L.H.M.

    1988-01-01

    The warfarin binding behaviour of a large tryptic fragment (residues 198–585 which comprise domains two and three) and of a large peptic fragment (residues 1–387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to

  15. A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

    DEFF Research Database (Denmark)

    Rasmussen, Trine Nygaard; Plenge, Per; Bay, Tina

    2009-01-01

    The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect t...

  16. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  17. Ecosystem processes and human influences regulate streamflow response to climate change at long-term ecological research sites

    Science.gov (United States)

    Julia A. Jones; Irena F. Creed; Kendra L. Hatcher; Robert J. Warren; Mary Beth Adams; Melinda H. Benson; Emery Boose; Warren A. Brown; John L. Campbell; Alan Covich; David W. Clow; Clifford N. Dahm; Kelly Elder; Chelcy R. Ford; Nancy B. Grimm; Donald L Henshaw; Kelli L. Larson; Evan S. Miles; Kathleen M. Miles; Stephen D. Sebestyen; Adam T. Spargo; Asa B. Stone; James M. Vose; Mark W. Williams

    2012-01-01

    Analyses of long-term records at 35 headwater basins in the United States and Canada indicate that climate change effects on streamflow are not as clear as might be expected, perhaps because of ecosystem processes and human influences. Evapotranspiration was higher than was predicted by temperature in water-surplus ecosystems and lower than was predicted in water-...

  18. [3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

    International Nuclear Information System (INIS)

    Marquis, O.; Robaut, C.; Cavero, I.

    1988-01-01

    In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

  19. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  20. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

    Directory of Open Access Journals (Sweden)

    Cecilia Herraiz

    Full Text Available The melanocortin 1 receptor gene (MC1R expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH. Human MC1R has an inefficient poly(A site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3. Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR might promote an isoform switch from canonical MC1R (MC1R-001 to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

  1. Primary structure of human pancreatic elastase 2 determined by sequence analysis of the cloned mRNA

    International Nuclear Information System (INIS)

    Fletcher, T.S.; Shen, W.F.; Largman, C.

    1987-01-01

    A cDNA encoding elastase 2 has been cloned from a human pancreatic cDNA library. The cDNA contains a translation initiation site and a poly(A) recognition site and encodes a protein of 269 amino acids, including a proposed 16-residue signal peptide. The amino acid sequence of the deduced mature protein contains a 12-residue activation peptide containing a cysteine at residue 1 similar to that of chymotryspin. The proposed active enzyme contains all of the characteristic active-site amino acids, including His-57, Asp-102, and Ser-195. The S1 binding pocket is bounded by Gly-216 and Ser-226, making this pocket intermediate in size between chymotrypsins and elastase 1 or protease E, consistent with the substrate specificity of elastase 2 for long-chain aliphatic or aromatic amino acids. Computer modeling studies using the amino acid sequence of elastase 2 superimposed on the X-ray structure of porcine elastase 1 suggest that a change of Gln-192 in elastase 1 to Asn-192 in elastase 2 may account for the lower catalytic efficiency of the latter enzyme. Several basic residues appear to be near the ends of the extended binding pocket of elastases which might serve to anchor the enzyme to the elastin substrate. These studies indicate that elastases 2 and elastase 1 both contain an Arg-65A as well as a basic dipeptide at 223/224 which is not present in chymotrypsins. In addition, Arg-217A is present in humaan elastase 2 but absent in rat pancreatic protein which has been proposed to be an elastase 2 on the basis of sequence homology, but which was not isolated during screening of rat pancreatic tissue extracts for elastolytic activity

  2. Final environmental impact statement for the Nevada test site and off-site locations in the State of Nevada. Human health risks and safety impacts study, Volume 1, Appendix H

    International Nuclear Information System (INIS)

    1996-08-01

    Proposed changes in the Nevada Test Site (NTS) operations, as well as the US Department of Energy (DOE) policy of reviewing sitewide National Environmental Policy Act (NEPA) documents, have resulted in the need for the US Department of Energy, Nevada Operations Office (DOE/NV) Operations Office to prepare a new Environmental Impact Statement (EIS) for the NTS. This report has been prepared to assess the human health and safety impacts from operations expected to be carried out under each of the four alternatives defined in the NTS EIS. These alternatives are: Alternative 1, Continue Current Operations (No Action); Alternative 2, Discontinue Operations; Alternative 3, Expanded Use; and Alternative 4, Alternate Use of Withdrawn Lands

  3. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  4. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

    International Nuclear Information System (INIS)

    Tate, K.M.; Higgins, D.L.; Holmes, W.E.; Winkler, M.E.; Heyneker, H.L.; Vehar, G.A.

    1987-01-01

    Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 → Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 → Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with → 2 -macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form

  5. Manipulation of the membrane binding site of vitamin K-dependent proteins: Enhanced biological function of human factor VII

    OpenAIRE

    Shah, Amit M.; Kisiel, Walter; Foster, Donald C.; Nelsestuen, Gary L.

    1998-01-01

    Recent studies suggested that modification of the membrane contact site of vitamin K-dependent proteins may enhance the membrane affinity and function of members of this protein family. The properties of a factor VII mutant, factor VII-Q10E32, relative to wild-type factor VII (VII, containing P10K32), have been compared. Membrane affinity of VII-Q10E32 was about 20-fold higher than that of wild-type factor VII. The rate of autoactivation VII-Q10E32 with soluble tissue factor was 100-fold fast...

  6. Human Health and Ecological Risk Assessment for the Operation of the Explosives Waste Treatment Facility at Site 300 of the Lawrence Livermore National Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Gallegos, G; Daniels, J; Wegrecki, A

    2007-10-01

    This document contains the human health and ecological risk assessment for the Resource Recovery and Conservation Act (RCRA) permit renewal for the Explosives Waste Treatment Facility (EWTF). Volume 1 is the text of the risk assessment, and Volume 2 (provided on a compact disc) is the supporting modeling data. The EWTF is operated by the Lawrence Livermore National Laboratory (LLNL) at Site 300, which is located in the foothills between the cities of Livermore and Tracy, approximately 17 miles east of Livermore and 8 miles southwest of Tracy. Figure 1 is a map of the San Francisco Bay Area, showing the location of Site 300 and other points of reference. One of the principal activities of Site 300 is to test what are known as 'high explosives' for nuclear weapons. These are the highly energetic materials that provide the force to drive fissionable material to criticality. LLNL scientists develop and test the explosives and the integrated non-nuclear components in support of the United States nuclear stockpile stewardship program as well as in support of conventional weapons and the aircraft, mining, oil exploration, and construction industries. Many Site 300 facilities are used in support of high explosives research. Some facilities are used in the chemical formulation of explosives; others are locations where explosive charges are mechanically pressed; others are locations where the materials are inspected radiographically for such defects as cracks and voids. Finally, some facilities are locations where the machined charges are assembled before they are sent to the onsite test firing facilities, and additional facilities are locations where materials are stored. Wastes generated from high-explosives research are treated by open burning (OB) and open detonation (OD). OB and OD treatments are necessary because they are the safest methods for treating explosives wastes generated at these facilities, and they eliminate the requirement for further handling

  7. A combined spectroscopic and molecular docking study on site selective binding interaction of Toluidine blue O with Human and Bovine serum albumins

    Energy Technology Data Exchange (ETDEWEB)

    Selva Sharma, Arumugam [Department of Chemistry, Bharathiar University, Coimbatore 641046 (India); Anandakumar, Shanmugam [Department of Bioinformatics, Bharathiar University, Coimbatore 641046 (India); Ilanchelian, Malaichamy, E-mail: chelian73@yahoo.com [Department of Chemistry, Bharathiar University, Coimbatore 641046 (India)

    2014-07-01

    In the present investigation the interaction of a biologically active photodynamic therapeutic agent Toluidine blue O (TBO) with Serum albumins viz Human serum albumin (HSA) and Bovine serum albumin (BSA) was studied using absorption, emission, circular dichroism spectroscopy and molecular docking experiments. The emission titration experiments between HSA/BSA and TBO revealed the existence of strong interactions between TBO and the proteins. The site competitive experiment of HSA and BSA showed that the primary binding site of TBO is located in site I of HSA/BSA involving hydrophobic, hydrogen bonding and electrostatic interaction. To ascertain the results of site competitive experiments, molecular docking was utilized to characterize the binding models of TBO–HSA/BSA complexes. From the molecular docking studies, free energy calculations were undertaken to examine the energy contributions and the role of various amino acid residues of HSA/BSA in TBO binding. The existence of Forster Resonance Energy Transfer (FRET) between the ligand and the protein was utilized to calculate the donor–acceptor distance of TBO and protein. The TBO induced conformational changes of HSA/BSA was established using synchronous emission, three dimensional emission and circular dichroism studies. - Highlights: • Site selective binding interaction of TBO with HSA and BSA were investigated. • TBO quenches the intrinsic fluorescence of HSA/BSA by static quenching process. • Computational studies of TBO with HSA/BSA substantiate the experimental findings. • 3D and CD spectral studies of TBO–HSA/BSA revealed structural changes in protein. • The distance (r) between TBO and HSA/BSA were estimated from FRET theory.

  8. Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat

    Directory of Open Access Journals (Sweden)

    Jin Hong

    2009-12-01

    Full Text Available Abstract Background The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain is its distant relative, α-dystrobrevin. The α-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands in the dystrophin glycoprotein complex. Results We show here that, contrary to the literature, most α-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse α-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms α-dystrobrevin-4 and -5 are present in most mammals but specifically ablated in mouse and rat. Conclusion Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the α-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the α-dystrobrevins (and therefore the dystrophin glycoprotein complexes of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.

  9. An integrated in silico approach to design specific inhibitors targeting human poly(a-specific ribonuclease.

    Directory of Open Access Journals (Sweden)

    Dimitrios Vlachakis

    Full Text Available Poly(A-specific ribonuclease (PARN is an exoribonuclease/deadenylase that degrades 3'-end poly(A tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARN's catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNP-poly(A inhibits PARN competitively. Our approach provides an efficient integrated platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential.

  10. Land and Water Use Characteristics and Human Health Input Parameters for use in Environmental Dosimetry and Risk Assessments at the Savannah River Site. 2016 Update

    Energy Technology Data Exchange (ETDEWEB)

    Jannik, G. Tim [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Hartman, Larry [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Stagich, Brooke [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2016-09-26

    Operations at the Savannah River Site (SRS) result in releases of small amounts of radioactive materials to the atmosphere and to the Savannah River. For regulatory compliance purposes, potential offsite radiological doses are estimated annually using computer models that follow U.S. Nuclear Regulatory Commission (NRC) regulatory guides. Within the regulatory guides, default values are provided for many of the dose model parameters, but the use of applicant site-specific values is encouraged. Detailed surveys of land-use and water-use parameters were conducted in 1991 and 2010. They are being updated in this report. These parameters include local characteristics of meat, milk and vegetable production; river recreational activities; and meat, milk and vegetable consumption rates, as well as other human usage parameters required in the SRS dosimetry models. In addition, the preferred elemental bioaccumulation factors and transfer factors (to be used in human health exposure calculations at SRS) are documented. The intent of this report is to establish a standardized source for these parameters that is up to date with existing data, and that is maintained via review of future-issued national references (to evaluate the need for changes as new information is released). These reviews will continue to be added to this document by revision.

  11. Land and Water Use Characteristics and Human Health Input Parameters for use in Environmental Dosimetry and Risk Assessments at the Savannah River Site 2017 Update

    Energy Technology Data Exchange (ETDEWEB)

    Jannik, T. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Stagich, B. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2017-05-25

    Operations at the Savannah River Site (SRS) result in releases of relatively small amounts of radioactive materials to the atmosphere and to the Savannah River. For regulatory compliance purposes, potential offsite radiological doses are estimated annually using computer models that follow U.S. Nuclear Regulatory Commission (NRC) regulatory guides. Within the regulatory guides, default values are provided for many of the dose model parameters, but the use of site-specific values is encouraged. Detailed surveys of land-use and water-use parameters were conducted in 1991, 2008, 2010, and 2016 and are being concurred with or updated in this report. These parameters include local characteristics of meat, milk, and vegetable production; river recreational activities; and meat, milk, and vegetable consumption rates, as well as other human usage parameters required in the SRS dosimetry models. In addition, the preferred elemental bioaccumulation factors and transfer factors (to be used in human health exposure calculations at SRS) are documented. The intent of this report is to establish a standardized source for these parameters that is up to date with existing data, and that is maintained via review of future-issued national references (to evaluate the need for changes as new information is released). These reviews will continue to be added to this document by revision.

  12. Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

    Energy Technology Data Exchange (ETDEWEB)

    Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.; Duellberg, Christian; Cade, Nicholas I.; Kleiner, Ralph E.; Forth, Scott; Surrey, Thomas; Nogales, Eva; Kapoor, Tarun M.

    2016-04-01

    The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

  13. G-Quadruplexes Involving Both Strands of Genomic DNA Are Highly Abundant and Colocalize with Functional Sites in the Human Genome.

    Directory of Open Access Journals (Sweden)

    Andrzej S Kudlicki

    Full Text Available The G-quadruplex is a non-canonical DNA structure biologically significant in DNA replication, transcription and telomere stability. To date, only G4s with all guanines originating from the same strand of DNA have been considered in the context of the human nuclear genome. Here, I discuss interstrand topological configurations of G-quadruplex DNA, consisting of guanines from both strands of genomic DNA; an algorithm is presented for predicting such structures. I have identified over 550,000 non-overlapping interstrand G-quadruplex forming sequences in the human genome--significantly more than intrastrand configurations. Functional analysis of interstrand G-quadruplex sites shows strong association with transcription initiation, the results are consistent with the XPB and XPD transcriptional helicases binding only to G-quadruplex DNA with interstrand topology. Interstrand quadruplexes are also enriched in origin of replication sites. Several topology classes of interstrand quadruplex-forming sequences are possible, and different topologies are enriched in different types of structural elements. The list of interstrand quadruplex forming sequences, and the computer program used for their prediction are available at the web address http://moment.utmb.edu/allquads.

  14. A review of environmental fate, body burdens, and human health risk assessment of PCDD/Fs at two typical electronic waste recycling sites in China

    International Nuclear Information System (INIS)

    Chan, Janet Kit Yan; Wong, Ming H.

    2013-01-01

    This paper reviews the levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in different environmental media, human body burdens and health risk assessment results at e-waste recycling sites in China. To provide an indication of the seriousness of the pollution levels in the e-waste recycling sites in China, the data are compared with guidelines and available existing data for other areas. The comparison clearly shows that PCDD/Fs derived from the recycling processes lead to serious pollution in different environmental compartments (such as air, soil, sediment, dust and biota) and heavy body burdens. Of all kinds of e-waste recycling operations, open burning of e-waste and acid leaching activities are identified as the major sources of PCDD/Fs. Deriving from the published data, the estimated total exposure doses via dietary intake, inhalation, soil/dust ingestion and dermal contact are calculated for adults, children and breast-fed infants living in two major e-waste processing locations in China. The values ranged from 5.59 to 105.16 pg WHO-TEQ/kg bw/day, exceeding the tolerable daily intakes recommended by the WHO (1–4 pg WHO-TEQ/kg bw/day). Dietary intake is the most important exposure route for infants, children and adults living in these sites, contributing 60–99% of the total intakes. Inhalation is the second major exposure route, accounted for 12–30% of the total exposure doses of children and adults. In order to protect the environment and human health, there is an urgent need to control and monitor the informal e-waste recycling operations. Knowledge gaps, such as comprehensive dietary exposure data, epidemiological and clinical studies, body burdens of infants and children, and kinetics about PCDD/Fs partitions among different human tissues should be addressed. - Highlights: ► PCDD/F levels at e-waste recycling sites in China were reviewed. ► Data on environment and body burden and health risk assessment results were reviewed

  15. A review of environmental fate, body burdens, and human health risk assessment of PCDD/Fs at two typical electronic waste recycling sites in China

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Janet Kit Yan, E-mail: chanjky@hku.hk [School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong (China); Wong, Ming H., E-mail: mhwong@hkbu.edu.hk [Croucher Institute for Environmental Sciences, and Department of Biology, Hong Kong Baptist University, Hong Kong (China)

    2013-10-01

    This paper reviews the levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in different environmental media, human body burdens and health risk assessment results at e-waste recycling sites in China. To provide an indication of the seriousness of the pollution levels in the e-waste recycling sites in China, the data are compared with guidelines and available existing data for other areas. The comparison clearly shows that PCDD/Fs derived from the recycling processes lead to serious pollution in different environmental compartments (such as air, soil, sediment, dust and biota) and heavy body burdens. Of all kinds of e-waste recycling operations, open burning of e-waste and acid leaching activities are identified as the major sources of PCDD/Fs. Deriving from the published data, the estimated total exposure doses via dietary intake, inhalation, soil/dust ingestion and dermal contact are calculated for adults, children and breast-fed infants living in two major e-waste processing locations in China. The values ranged from 5.59 to 105.16 pg WHO-TEQ/kg bw/day, exceeding the tolerable daily intakes recommended by the WHO (1–4 pg WHO-TEQ/kg bw/day). Dietary intake is the most important exposure route for infants, children and adults living in these sites, contributing 60–99% of the total intakes. Inhalation is the second major exposure route, accounted for 12–30% of the total exposure doses of children and adults. In order to protect the environment and human health, there is an urgent need to control and monitor the informal e-waste recycling operations. Knowledge gaps, such as comprehensive dietary exposure data, epidemiological and clinical studies, body burdens of infants and children, and kinetics about PCDD/Fs partitions among different human tissues should be addressed. - Highlights: ► PCDD/F levels at e-waste recycling sites in China were reviewed. ► Data on environment and body burden and health risk assessment results were reviewed

  16. 5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mrna: a potential common mechanism for tombusviridae.

    Science.gov (United States)

    Fabian, Marc R; White, K Andrew

    2004-07-09

    Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5' cap and a 3' poly(A) tail and instead contains a 3'-terminal RNA sequence that acts as a cap-independent translational enhancer (3' CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3' CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3' CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base pairing with another SL, SL3, in the 5' untranslated region of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5'-3' RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5' untranslated regions and the 3' CITE, and both genomic and subgenomic 5'-3' interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5'-3' RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.

  17. Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

    Directory of Open Access Journals (Sweden)

    Jianjun He

    Full Text Available It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

  18. The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.

    Science.gov (United States)

    Hountondji, Codjo; Bulygin, Konstantin; Créchet, Jean-Bernard; Woisard, Anne; Tuffery, Pierre; Nakayama, Jun-Ichi; Frolova, Ludmila; Nierhaus, Knud H; Karpova, Galina; Baouz, Soria

    2014-01-01

    We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

  19. Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

    Science.gov (United States)

    Tausendschön, Michaela; Rehli, Michael; Dehne, Nathalie; Schmidl, Christian; Döring, Claudia; Hansmann, Martin-Leo; Brüne, Bernhard

    2015-01-01

    Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

    International Nuclear Information System (INIS)

    Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

    1989-01-01

    The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

  1. Synthesis, purification, and characterization of an Arg152 → Glu site-directed mutant of recombinant human blood clotting factor VII

    International Nuclear Information System (INIS)

    Wildgoose, P.; Kisiel, W.; Berkner, K.L.

    1990-01-01

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg 152 -Ile 153 . Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg 152 → Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M r ∼40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX

  2. Synthesis, purification, and characterization of an Arg sub 152 yields Glu site-directed mutant of recombinant human blood clotting factor VII

    Energy Technology Data Exchange (ETDEWEB)

    Wildgoose, P.; Kisiel, W. (Univ. of New Mexico, Albuquerque (USA)); Berkner, K.L. (ZymoGenetics, Inc., Seattle, WA (USA))

    1990-04-03

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg{sub 152}-Ile{sub 153}. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg{sub 152} {yields} Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M{sup r}{approx}40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX.

  3. Sites of origin of oral cavity cancer in nonsmokers vs smokers: possible evidence of dental trauma carcinogenesis and its importance compared with human papillomavirus.

    Science.gov (United States)

    Perry, Brendan J; Zammit, Andrew P; Lewandowski, Andrew W; Bashford, Julia J; Dragovic, Adrian S; Perry, Emily J; Hayatbakhsh, Reza; Perry, Christopher F L

    2015-01-01

    The relatively high and possibly rising incidence of mouth squamous cell carcinoma in nonsmokers, especially women, without obvious cause has been noted by previous authors. Is chronic dental trauma and irritation a carcinogen, and what is its importance compared with human papillomavirus (HPV) oropharyngeal cancer in nonsmokers? To determine whether oral cavity cancers occurred more commonly at sites of dental trauma and how the position of these cancers varied between nonsmokers lacking major identified carcinogens and smokers. If these cancers occurred more frequently at sites of chronic trauma, especially in nonsmokers, it would suggest chronic dental trauma as a possible carcinogen. A retrospective analysis of 881 patients with oral cavity or oropharyngeal cancers seen through a tertiary referral hospital between 2001 and 2011 was performed. Patient medical records were analyzed to determine the location of the tumor within the oral cavity and oropharynx and how it relates to patient demographics, smoking and alcohol histories, and comorbidities. Dental histories were also sought, including use of dentures. Nonsmokers comprised 87 of 390 patients with mouth cancer (22%) and 48 of 334 patients with oropharyngeal cancer (14%). Female nonsmoking patients included 53 with oral cancer (61%) but only 12 with oropharyngeal squamous cell carcinoma (25%). Oral cancers occurred on the lateral tongue, a potential site of chronic dental trauma, in 57 nonsmokers (66%) compared with 107 smokers/ex-smokers (33%) (P Oral cavity cancers occur predominantly at sites of potential dental and denture trauma, especially in nonsmokers without other risk factors. Recognizing teeth irritation as a potential carcinogen would have an impact on prevention and treatment strategies.

  4. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    International Nuclear Information System (INIS)

    Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

    2011-01-01

    Highlights: → Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). → Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. → HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  5. Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

    Science.gov (United States)

    Jia, Pingping; Chastain, Megan; Zou, Ying; Her, Chengtao

    2017-01-01

    Abstract Aberrant formation of interstitial telomeric sequences (ITSs) promotes genome instabilities. However, it is unclear how aberrant ITS formation is suppressed in human cells. Here, we report that MLH1, a key protein involved in mismatch repair (MMR), suppresses telomeric sequence insertion (TSI) at intra-chromosomal regions. The frequency of TSI can be elevated by double-strand break (DSB) inducer and abolished by ATM/ATR inhibition. Suppression of TSI requires MLH1 recruitment to DSBs, indicating that MLH1's role in DSB response/repair is important for suppressing TSI. Moreover, TSI requires telomerase activity but is independent of the functional status of p53 and Rb. Lastly, we show that TSI is associated with chromosome instabilities including chromosome loss, micronuclei formation and chromosome breakage that are further elevated by replication stress. Our studies uncover a novel link between MLH1, telomerase, telomere and genome stability. PMID:28180301

  6. Exposure to different toxic chemicals: a threat to environment and human health in mining sites in Tanzania

    International Nuclear Information System (INIS)

    Magduala, J.J.

    2009-01-01

    The mining activities in Tanzania have been existed since time immemorial whereby traditional mining was practiced. However until now the country is still endowed with abundant mineral resources including gold, tanzanite diamonds, iron ore, salt, gypsum, gemstones, natural gas, phosphate, coal, cobalt and nickel. The country's major gold fields are located in Geita, Musoma, Tarime, Chunya and Mpanda. During the last decade, local and foreign investors intensified their mining activities in Tanzania. This resulted in increased use of hazardous chemicals like mercury and cyanide which are harmful and toxic. In this report, the extent and impact to long term exposure of such chemicals to both natural environment and animals including human beings will be discussed. Recommendations to local and international investors and policy markers regarding the safe and sustainable use of harmful chemicals will also be discussed.(author)

  7. Different Phenotypes in Human Prostate Cancer: α6 or α3 Integrin in Cell-extracellular Adhesion Sites

    Directory of Open Access Journals (Sweden)

    Monika Schmelz

    2002-01-01

    Full Text Available The distribution of α6/α3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized α6/α3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The α6/α3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA. The associations were assessed by statistical methods. Eighty percent of the tumors expressed the α6 or α3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both α6 and α3 subunits, type II exclusively expressed a6 integrin, and type III expressed α3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The α6/α3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, α6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the α6 subunit did not colocalize with the α3 subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the α6β1 and/or α3β1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.

  8. Late Neolithic phytolith and charcoal records of human activities and vegetation change in Shijiahe culture, Tanjialing site, China.

    Directory of Open Access Journals (Sweden)

    Xiao Hong Zhu

    Full Text Available There is significant archaeological evidence marking the collapse of the Shijiahe culture in the middle reaches of the Yangtze River in China during the late Neolithic Period. However, the causes for this cultural collapse remain unclear. Our sedimentary records from a 3.3 m long profile and 76 phytolith and charcoal samples from the Tanjialing archaeological sites provide records of interactions between an ancient culture and vegetation change. During the early Shijiahe culture (c, 4850-4400 cal BP, the climate was warm and humid. Fire was intensively used to clear the vegetation. In the mid-period of the Shijiahe culture (c, 4400-4200 cal BP, the climate became slightly dry-cold and this was accompanied by decreasing water, leading to settlements. From c, 4200 cal BP, severe drought eroded the economic foundation of rice-cultivation. These conditions forced people to abandon the Shijiahe ancient city to find water in other regions, leading to the collapse of the Shijiahe culture.

  9. Modulation of salivation and heartburn in response to the site of acid infusion in the human oesophagus.

    Science.gov (United States)

    Dutta, S K; Agrawal, K; Mahmoud, M A

    2010-09-01

    The pathogenesis of gastro-oesophageal reflux disease includes increased acid reflux, reduced salivation and impaired peristalsis. This may depend upon the height of acid wave and magnitude of oesophageal mucosal exposure. Interestingly, the effect of site of acid infusion upon salivary secretion and heartburn has not been examined in any detail. To examine whether acid infusion in the upper oesophagus may cause increased salivation and heartburn as compared with acid infusion in the lower oesophagus. Twelve healthy male subjects (mean age 30) received infusions of HCl, citric acid and acetic acid at 10 and 20 cm above the lower oesophageal sphincter (LES) for fixed time periods. Parotid saliva collected periodically and heartburn severity scored using standardized scale. Standard statistical methods (paired t-tests, analysis of variance) were used to determine the significance of results. Acid infusion in the upper oesophagus increased parotid flow rate as compared with that in the lower oesophagus (P heartburn score at 20 cm as well as 10 cm above LES (P heartburn in response to acid infusion in the upper vs. lower part of the oesophagus. 2010 Blackwell Publishing Ltd.

  10. Agricultural land use and human presence around breeding sites increase stress-hormone levels and decrease body mass in barn owl nestlings.

    Science.gov (United States)

    Almasi, Bettina; Béziers, Paul; Roulin, Alexandre; Jenni, Lukas

    2015-09-01

    Human activities can have a suite of positive and negative effects on animals and thus can affect various life history parameters. Human presence and agricultural practice can be perceived as stressors to which animals react with the secretion of glucocorticoids. The acute short-term secretion of glucocorticoids is considered beneficial and helps an animal to redirect energy and behaviour to cope with a critical situation. However, a long-term increase of glucocorticoids can impair e.g. growth and immune functions. We investigated how nestling barn owls (Tyto alba) are affected by the surrounding landscape and by human activities around their nest sites. We studied these effects on two response levels: (a) the physiological level of the hypothalamus-pituitary-adrenal axis, represented by baseline concentrations of corticosterone and the concentration attained by a standardized stressor; (b) fitness parameters: growth of the nestlings and breeding performance. Nestlings growing up in intensively cultivated areas showed increased baseline corticosterone levels late in the season and had an increased corticosterone release after a stressful event, while their body mass was decreased. Nestlings experiencing frequent anthropogenic disturbance had elevated baseline corticosterone levels, an increased corticosterone stress response and a lower body mass. Finally, breeding performance was better in structurally more diverse landscapes. In conclusion, anthropogenic disturbance affects offspring quality rather than quantity, whereas agricultural practices affect both life history traits.

  11. Human bone hardness seems to depend on tissue type but not on anatomical site in the long bones of an old subject.

    Science.gov (United States)

    Ohman, Caroline; Zwierzak, Iwona; Baleani, Massimiliano; Viceconti, Marco

    2013-02-01

    It has been hypothesised that among different human subjects, the bone tissue quality varies as a function of the bone segment morphology. The aim of this study was to assess and compare the quality, evaluated in terms of hardness of packages of lamellae, of cortical and trabecular bones, at different anatomical sites within the human skeleton. The contralateral six long bones of an old human subject were indented at different levels along the diaphysis and at both epiphyses of each bone. Hardness value, which is correlated to the degree of mineralisation, of both cortical and trabecular bone tissues was calculated for each indentation location. It was found that the cortical bone tissue was harder (+18%) than the trabecular one. In general, the bone hardness was found to be locally highly heterogeneous. In fact, considering one single slice obtained for a bone segment, the coefficient of variation of the hardness values was up to 12% for cortical bone and up to 17% for trabecular bone. However, the tissue hardness was on average quite homogeneous within and among the long bones of the studied donor, although differences up to 9% among levels and up to 7% among bone segments were found. These findings seem not to support the mentioned hypothesis, at least not for the long bones of an old subject.

  12. mRNA related to insulin family in human placenta

    International Nuclear Information System (INIS)

    Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-01-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

  13. Identification of a negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Ryan E Pavlovicz

    Full Text Available Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs. These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological

  14. CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites.

    Science.gov (United States)

    Baker, K K; Levine, M M; Morison, J; Phillips, A; Barry, E M

    2009-05-01

    Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype. In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen. © 2009 Blackwell Publishing Ltd.

  15. Expression of c-myc and c-fos and binding sites for estradiol and progesterone in human pituitary tumors.

    Science.gov (United States)

    Machiavelli, G A; Rivolta, C M; Artese, R; Basso, A; Burdman, J A

    1998-12-01

    We studied the concentration of mRNA from the oncogenes c-myc and c-fos in human pituitary adenomas by Northern blot hybridization (35 somatotrophinomas, 9 prolactinomas, 21 nonsecreting and 3 adrenocorticotrophinomas). The concentration of estrogens and progesterone receptors was also investigated. The levels of c-myc and c-fos mRNA was higher in nonsecreting tumors which were generally the largest and had a higher percentage of recurrence after surgery than the other groups. High concentration of estrogen receptors was observed in tumors derived from cells which are normally the target of this hormone, mainly prolactinomas. They were also present in somatotrophic and nonsecreting adenomas, related to the presence of prolactin or gonadotrophin cells in these tumors. The presence of estrogen receptors indicates that the tumor cells maintain their differentiation and a good prognosis as is the case for prolactinomas. We did not find any relationship between estrogen receptors and the concentration of c-myc and c-fos oncogenes. Larger adenomas (mainly nonsecreting) had higher levels of c-myc and c-fos mRNA than the other tumors and they had an important percentage of recurrence after surgery. It is clear that tumor size is related to the outcome after surgery and that nonsecreting adenomas are usually large because of the late diagnosis. However two large somatotrophinomas with extrasellar expansion also had overexpression of both oncogenes and both relapsed after surgery.

  16. A multi-site array for combined local electrochemistry and electrophysiology in the non-human primate brain.

    Science.gov (United States)

    Disney, Anita A; McKinney, Collin; Grissom, Larry; Lu, Xuekun; Reynolds, John H

    2015-11-30

    Currently, the primary technique employed in circuit-level study of the brain is electrophysiology, recording local field or action potentials (LFPs or APs). However most communication between neurons is chemical and the relationship between electrical activity within neurons and chemical signaling between them is not well understood in vivo, particularly for molecules that signal at least in part by non-synaptic transmission. We describe a multi-contact array and accompanying head stage circuit that together enable concurrent electrophysiological and electrochemical recording. The array is small (electrochemistry) recording. This system is designed for concurrent, dual-mode recording. It is also the only system designed explicitly to meet the challenges of recording in non-human primates. Our system offers the possibility for conducting in vivo studies in a range of species that examine the relationship between the electrical activity of neurons and their chemical environment, with exquisite spatial and temporal precision. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  17. The effectiveness of search dogs compared with humans in searching difficult terrain at turbine sites for bat fatalities

    Energy Technology Data Exchange (ETDEWEB)

    Mathews, Fiona

    2011-07-01

    Full text: Many wind farms in the UK and elsewhere in northern Europe are situated in habitat with dense tall vegetation such as arable fields and upland heaths. This makes surveying for bat fatalities extremely difficult. To facilitate a multi-centre study of the effects of wind turbines on British bats, we have therefore conducted controlled trials of the relative success of trained search dogs and ecologists in retrieving bat carcasses. Although dogs have been used previously in ecological surveys for bats, this is the first time they have been specifically trained for use in 'difficult to survey' habitats. Two ecologists and two Labrador dogs with handlers were each given the opportunity to retrieve up to 45 bat carcasses in a range of habitat types. Their efficiency in terms of overall search time, costs, and retrieval abilities were evaluated. Our results indicate that high rates of retrieval can be achieved by dogs, even in dense vegetation up to 75cm high. Further, a typical 100m2 search area can be surveyed in less than half the time taken by humans. The limitations of using search dogs, and their ability to detect the presence of bats that have been scavenged are also presented (presentation supported with video footage). (Author)

  18. Study on the radiation dose assessment of non-human species near a site in the south of China

    International Nuclear Information System (INIS)

    Li Jianguo; Han Baohua; Ma Binghui; Han Xuelei; Shang Honglian; Huang Peng; Geng Li

    2012-01-01

    In the external environment of a site located in the South China, the Chinese toad (Bufo gargarizans), dry duck and teal, and pine tree (Pinus massoniana Lamb) are studied as reference organisms. Through investigation, the parameters of biological characteristics of the species are provided. The radionuclide concentration ratio CR (defined as the ratio of activity concentration in biota whole body (Bq · kg -1 fresh weight) and activity concentration of soil (Bq · kg -1 dry weight) or activity concentration of filtered water(Bq · L -1 )) are provided by collecting biological and environmental media samples and 90 Sr and 137 Cs activity analysis. By the ERICA tool of EC, the dose rates of the species for 90 Sr and 137 Cs are estimated. The results show that the dose rates of 90 Sr and 137 Cs for Chinese toad are 1.69 E 03 μGy · h -1 and 6.96 E-03 μGy · h -1 respectively, the total dose rate is 8.65 E-03 μGy · h -1 . The dose rates of 90 Sr and 137 Cs for teal are 1.56 E-04 μGy · h -1 and 2.68 E-04EμGy · h -1 , the total dose rate is 4.24E-04 μGy · h -1 . The dose rates of 90 Sr and 137 Cs for dry duck are 2.04E-04 μGy · h -1 and 2.29 E-04EμGy · h -1 , the total dose rate is 4.33E-04 μGy · h -1 . The total dose rates of the reference animals are well below the IAEA recommended dose limit for terrestrial animals (40 μGy · h -1 ). The order of dose rates of 90 Sr and 137 Cs for pine, from high to low are: pine needles > pine branches > pine trunk. The total dose rate of pine needles is 1.38E-03 μGy · h -1 , which is well below the IAEA recommended dose limit for terrestrial plants (400 μGy · h -1 ). (authors)

  19. Human Activity and Habitat Characteristics Influence Shorebird Habitat Use and Behavior at a Vancouver Island Migratory Stopover Site.

    Science.gov (United States)

    Murchison, Colleen R; Zharikov, Yuri; Nol, Erica

    2016-09-01

    Pacific Rim National Park Reserve on Vancouver Island, British Columbia, Canada, has 16 km of coastal beaches that attract many thousands of people and shorebirds (S.O. Charadrii) every year. To identify locations where shorebirds concentrate and to determine the impact of human activity and habitat characteristics on shorebirds, we conducted shorebird and visitor surveys at 20 beach sectors (across 20 total km of beach) during fall migration in 2011-2014 and spring migration in 2012 and 2013. Using zero-inflated negative binomial regression and a model selection approach, we found that beach width and number of people influenced shorebird use of beach sectors (Bayesian information criterion weight of top model = 0.69). Shorebird absence from beaches was associated with increasing number of people (parameter estimate from top model: 0.38; 95 % CI 0.19, 0.57) and decreasing beach width (parameter estimate: -0.32; 95 % CI -0.47, -0.17). Shorebirds spent more time at wider beaches (parameter estimate: 0.68; 95 % CI 0.49, 0.87). Close proximity to people increased the proportion of time shorebirds spent moving, while shorebirds spent more time moving and less time foraging on wider beaches than on narrower ones. Shorebird disturbance increased with proximity of people, activity speed, and presence of dogs. Based on our findings, management options, for reducing shorebird disturbance at Pacific Rim National Park Reserve and similar shorebird stopover areas, include mandatory buffer distances between people and shorebirds, restrictions on fast-moving activities (e.g., running, biking), prohibiting dogs, and seasonal closures of wide beach sections.

  20. Methodology and parameters for assessing human health effects for waste sites at the Savannah River Plant: Environmental information document

    International Nuclear Information System (INIS)

    King, C.M.; Marter, W.L.; Looney, B.B.; Pickett, J.B.

    1987-03-01

    This report provides a summary of the components of risk assessment and presents the technical basis for application of the risk evaluation process to the principal pollutants at SRP: radionuclides, toxic chemicals, and carcinogenic compounds. An extensive technical data base from the fields of radiation health physics, toxicology, and environmental sciences is required to accomplish this task. The origin and meaning of this data base is summarized for each class of contaminant and parameter values provided for use in numerical analysis of risk. The process of risk assessment is associated with uncertainties, a fact which is frequently stated in the technical literature addressing this subject. A review of risk assessment uncertainties and the limitations of predictive risk assessment are summarized. Risk estimators for each class of contaminants at the SRP have been tabulated for radionuclides, toxic chemicals, and carcinogens from the technical literature. Estimation of human health risk is not an additive process for radiation effects and chemical carcinogenesis since their respective dosimetric models are distinctly different even though the induction of cancer is reported to be the common end result. It is recommended in this report that risk estimation for radionuclides and chemical carcinogens should be tabulated separately and this recommendation has been applied in all environmental information documentation published by the Savannah River Laboratory. Impacts due to toxic chemicals in the biosphere should also be estimated as a separate entity since toxic chemical risk estimators are uniquely different and do not reflect the probability of a detrimental health effect. 23 refs., 4 figs., 13 tabs

  1. Sediment sequence and site formation processes at the Arbreda Cave, NE Iberian Peninsula, and implications on human occupation and climate change during the Last Glacial

    Directory of Open Access Journals (Sweden)

    M. Kehl

    2014-09-01

    Full Text Available The Arbreda Cave provides a detailed archaeological record of the Middle to Upper Palaeolithic and is a key site