Telomeric repeat factor 1 protein levels correlates with telomere length in colorectal cancer Los niveles proteicos del factor de repetición telomérico 1 se correlacionan con la longitud del telómero en el cáncer colorrectal

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Cristina Valls-Bautista

2012-11-01

Full Text Available Background: colorectal cancer is the third cancer cause of death in Spain. It is important to investigate new tumoral markers for early diagnosis, disease monitoring and prevention strategies. Telomeres protect the chromosome from degradation by nucleases and end-to-end fusion. The progressive loss of the telomeric ends of chromosomes is an important mechanism in the timing of human cellular aging. Telomeric Repeat Factor 1 (TRF1 is a protein that binds at telomere ends. Purpose: to measure the concentrations of TRF1 and the relationships among telomere length, telomerase activity, and TRF1 levels in tumor and normal colorectal mucosa. Method: from normal and tumoral samples of 83 patients who underwent surgery for colorectal cancer we analyzed TRF1 protein concentration by Western Blot, telomerase activity, by the fluorescent-telomeric repeat amplification protocol assay and telomere length by Southern Blot. Results: high levels of TRF1 were observed in 68.7% of tumor samples, while the majority of normal samples (59% showed negative or weak TRF1 concentrations. Among the tumor samples, telomere length was significantly associated with TRF1 protein levels (p = 0.023. Conclusions: a relationship was found between telomere length and TRF1 abundance protein in tumor samples, which means that TRF1 is an important factor in the tumor progression and maybe a diagnostic factor.

Caspase-Dependent Apoptosis Induced by Telomere Cleavage and TRF2 Loss

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Asha S. Multani

2000-07-01

Full Text Available Chromosomal abnormalities involving telomeric associations (TAs often precede replicative senescence and abnormal chromosome configurations. We report here that telomere cleavage following exposure to proapoptotic agents is an early event in apoptosis. Exposure of human and murine cancer cells to a variety of pro-apoptotic stimuli (staurosporine, thapsigargin, anti-Fas antibody, cancer chemotherapeutic agents resulted in telomere cleavage and aggregation, finally their extrusion from the nuclei. Telomere loss was associated with arrest of cells in G2/M phase and preceded DNA fragmentation. Telomere erosion and subsequent large-scale chromatin cleavage were inhibited by overexpression of the anti -apoptotic protein, bcl-2, two peptide caspase inhibitors (BACMK and zVADfmk, indicating that both events are regulated by caspase activation. The results demonstrate that telomere cleavage is an early chromatin alteration detected in various cancer cell lines leading to drug-induced apoptosis, suggest that this event contributes to mitotic catastrophe and induction of cell death. Results also suggest that the decrease of telomeric-repeat binding factor 2 (TRF2 may be the earliest event in the ara-C-induced telomere shortening, induction of endoreduplication and chromosomal fragmentation leading to cell death.

A generic Transcriptomics Reporting Framework (TRF) for 'omics data processing and analysis.

Science.gov (United States)

Gant, Timothy W; Sauer, Ursula G; Zhang, Shu-Dong; Chorley, Brian N; Hackermüller, Jörg; Perdichizzi, Stefania; Tollefsen, Knut E; van Ravenzwaay, Ben; Yauk, Carole; Tong, Weida; Poole, Alan

2017-12-01

A generic Transcriptomics Reporting Framework (TRF) is presented that lists parameters that should be reported in 'omics studies used in a regulatory context. The TRF encompasses the processes from transcriptome profiling from data generation to a processed list of differentially expressed genes (DEGs) ready for interpretation. Included within the TRF is a reference baseline analysis (RBA) that encompasses raw data selection; data normalisation; recognition of outliers; and statistical analysis. The TRF itself does not dictate the methodology for data processing, but deals with what should be reported. Its principles are also applicable to sequencing data and other 'omics. In contrast, the RBA specifies a simple data processing and analysis methodology that is designed to provide a comparison point for other approaches and is exemplified here by a case study. By providing transparency on the steps applied during 'omics data processing and analysis, the TRF will increase confidence processing of 'omics data, and regulatory use. Applicability of the TRF is ensured by its simplicity and generality. The TRF can be applied to all types of regulatory 'omics studies, and it can be executed using different commonly available software tools. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

The C-terminal extension unique to the long isoform of the shelterin component TIN2 enhances its interaction with TRF2 in a phosphorylation- and dyskeratosis congenita-cluster-dependent fashion.

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Nelson, Nya D; Dodson, Lois M; Escudero, Laura; Sukumar, Ann T; Williams, Christopher L; Mihalek, Ivana; Baldan, Alessandro; Baird, Duncan M; Bertuch, Alison A

2018-03-26

TIN2 is central to the shelterin complex, linking the telomeric proteins TRF1 and TRF2 with TPP1/POT1. Mutations in TINF2 , which encodes TIN2, that are found in dyskeratosis congenita (DC) result in very short telomeres and cluster in a region shared by the two TIN2 isoforms, TIN2S (short) and TIN2L (long). Here we show that TIN2L, but not TIN2S, is phosphorylated. TRF2 interacts more with TIN2L than TIN2S, and both the DC-cluster and phosphorylation promote this enhanced interaction. The binding of TIN2L, but not TIN2S, is affected by TRF2-F120, which is also required for TRF2's interaction with end processing factors such as Apollo. Conversely, TRF1 interacts more with TIN2S than with TIN2L. A DC-associated mutation further reduces TIN2L-TRF1, but not TIN2S-TRF1, interaction. Cells overexpressing TIN2L or phosphomimetic-TIN2L are permissive to telomere elongation, whereas cells overexpressing TIN2S or phosphodead-TIN2L are not. Telomere lengths are unchanged in cell lines in which TIN2L expression has been eliminated by CRISPR/Cas9-mediated mutation. These results indicate that TIN2 isoforms are biochemically and functionally distinguishable, and that shelterin composition could be fundamentally altered in patients with TINF2 mutations. Copyright © 2018 Nelson et al.

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

DEFF Research Database (Denmark)

Peng, Xu; Brügger, Kim; Shen, Biao

2003-01-01

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

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

International Nuclear Information System (INIS)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

2004-01-01

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

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Kenchappa, Chandra; Heiðarsson, Pétur Orri; Kragelund, Birthe

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds...... specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal...... in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2(Hb) through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2...

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein*

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Grüning, Clara S. R.; Mirecka, Ewa A.; Klein, Antonia N.; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F.; Stoldt, Matthias; Hoyer, Wolfgang

2014-01-01

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. PMID:24966331

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

International Nuclear Information System (INIS)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun; Kim, Eunhee; Cheong, Chaejoon; Cho, Myeon Haeng; Lee, Weontae

2014-01-01

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2 1–64 ) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2 1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences

The SpTransformer Gene Family (Formerly Sp185/333) in the Purple Sea Urchin and the Functional Diversity of the Anti-Pathogen rSpTransformer-E1 Protein

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Smith, L. Courtney; Lun, Cheng Man

2017-01-01

The complex innate immune system of sea urchins is underpinned by several multigene families including the SpTransformer family (SpTrf; formerly Sp185/333) with estimates of ~50 members, although the family size is likely variable among individuals of Strongylocentrotus purpuratus. The genes are small with similar structure, are tightly clustered, and have several types of repeats in the second of two exons and that surround each gene. The density of repeats suggests that the genes are positioned within regions of genomic instability, which may be required to drive sequence diversification. The second exon encodes the mature protein and is composed of blocks of sequence called elements that are present in mosaics of defined element patterns and are the major source of sequence diversity. The SpTrf genes respond swiftly to immune challenge, but only a single gene is expressed per phagocyte. Many of the mRNAs appear to be edited and encode proteins with altered and/or missense sequence that are often truncated, of which some may be functional. The standard SpTrf protein structure is an N-terminal glycine-rich region, a central RGD motif, a histidine-rich region, and a C-terminal region. Function is predicted from a recombinant protein, rSpTransformer-E1 (rSpTrf-E1), which binds to Vibrio and Saccharomyces, but not to Bacillus, and binds tightly to lipopolysaccharide, β-1,3-glucan, and flagellin, but not to peptidoglycan. rSpTrf-E1 is intrinsically disordered but transforms to α helical structure in the presence of binding targets including lipopolysaccharide, which may underpin the characteristics of binding to multiple targets. SpTrf proteins associate with coelomocyte membranes, and rSpTrf-E1 binds specifically to phosphatidic acid (PA). When rSpTrf-E1 is bound to PA in liposome membranes, it induces morphological changes in liposomes that correlate with PA clustering and leakage of luminal contents, and it extracts or removes PA from the bilayer. The

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

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de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-11-16

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Telomere length modulation in human astroglial brain tumors.

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Domenico La Torre

Full Text Available BACKGROUND: Telomeres alteration during carcinogenesis and tumor progression has been described in several cancer types. Telomeres length is stabilized by telomerase (h-TERT and controlled by several proteins that protect telomere integrity, such as the Telomere Repeat-binding Factor (TRF 1 and 2 and the tankyrase-poli-ADP-ribose polymerase (TANKs-PARP complex. OBJECTIVE: To investigate telomere dysfunction in astroglial brain tumors we analyzed telomeres length, telomerase activity and the expression of a panel of genes controlling the length and structure of telomeres in tissue samples obtained in vivo from astroglial brain tumors with different grade of malignancy. MATERIALS AND METHODS: Eight Low Grade Astrocytomas (LGA, 11 Anaplastic Astrocytomas (AA and 11 Glioblastoma Multiforme (GBM samples were analyzed. Three samples of normal brain tissue (NBT were used as controls. Telomeres length was assessed through Southern Blotting. Telomerase activity was evaluated by a telomere repeat amplification protocol (TRAP assay. The expression levels of TRF1, TRF2, h-TERT and TANKs-PARP complex were determined through Immunoblotting and RT-PCR. RESULTS: LGA were featured by an up-regulation of TRF1 and 2 and by shorter telomeres. Conversely, AA and GBM were featured by a down-regulation of TRF1 and 2 and an up-regulation of both telomerase and TANKs-PARP complex. CONCLUSIONS: In human astroglial brain tumours, up-regulation of TRF1 and TRF2 occurs in the early stages of carcinogenesis determining telomeres shortening and genomic instability. In a later stage, up-regulation of PARP-TANKs and telomerase activation may occur together with an ADP-ribosylation of TRF1, causing a reduced ability to bind telomeric DNA, telomeres elongation and tumor malignant progression.

FR-like EBNA1 binding repeats in the human genome

International Nuclear Information System (INIS)

D'Herouel, Aymeric Fouquier; Birgersdotter, Anna; Werner, Maria

2010-01-01

Epstein-Barr virus (EBV) is widely spread in the human population. EBV nuclear antigen 1 (EBNA1) is a transcription factor that activates viral genes and is necessary for viral replication and partitioning, which binds the EBV genome cooperatively. We identify similar EBNA1 repeat binding sites in the human genome using a nearest-neighbor positional weight matrix. Previously experimentally verified EBNA1 sites in the human genome are successfully recovered by our approach. Most importantly, 40 novel regions are identified in the human genome, constituted of tandemly repeated binding sites for EBNA1. Genes located in the vicinity of these regions are presented as possible targets for EBNA1-mediated regulation. Among these, four are discussed in more detail: IQCB1, IMPG1, IRF2BP2 and TPO. Incorporating the cooperative actions of EBNA1 is essential when identifying regulatory regions in the human genome and we believe the findings presented here are highly valuable for the understanding of EBV-induced phenotypic changes.

C-terminal low-complexity sequence repeats of Mycobacterium smegmatis Ku modulate DNA binding.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-01-24

Ku protein is an integral component of the NHEJ (non-homologous end-joining) pathway of DSB (double-strand break) repair. Both eukaryotic and prokaryotic Ku homologues have been characterized and shown to bind DNA ends. A unique feature of Mycobacterium smegmatis Ku is its basic C-terminal tail that contains several lysine-rich low-complexity PAKKA repeats that are absent from homologues encoded by obligate parasitic mycobacteria. Such PAKKA repeats are also characteristic of mycobacterial Hlp (histone-like protein) for which they have been shown to confer the ability to appose DNA ends. Unexpectedly, removal of the lysine-rich extension enhances DNA-binding affinity, but an interaction between DNA and the PAKKA repeats is indicated by the observation that only full-length Ku forms multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer. The C-terminal extension promotes DNA end-joining by T4 DNA ligase, suggesting that the PAKKA repeats also contribute to efficient end-joining. We suggest that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins.

Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer

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HU JIAN

2009-01-01

Full Text Available Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1 in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC. The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS and nuclear export signal (NES are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30 patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2014-09-26

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

DNA damage response and role of shelterin complex in human peripheral blood mononuclear cells exposed to gamma radiation

International Nuclear Information System (INIS)

Saini, Divyalakshmi; Das, Birajalaxmi

2013-01-01

Telomeres are the DNA protein structures that cap the ends of linear DNA. It consists of short repetitive DNA sequences (TTAGGG)n and specialized telomere binding proteins. There are six telomeric proteins (TRF1, TRF2, TIN2, TERF2, PTOP and POT1) called as shelterin complex/telosome which maintains telomere integrity. The function of this 'telosome' is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks, thereby preventing chromosome end-to-end fusions. DNA Damage Response (DDR) induced by radiation and its interaction with telomeric protein complex is poorly understood in human PBMCs at G 0 stage. Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome, which may lead to senescence or apoptosis. Ionizing radiation which induces a plethora of DNA lesions in human cell may also alter the expression of telomere associated proteins. In the present study, we have made an attempt to study the DNA damage response of telomere proteins in human peripheral blood mononuclear cells exposed to gamma radiation. Venous blood samples were collected from eight random healthy volunteers and PBMCs were separated. Dose response as well as time point kinetics study was carried out at transcription as well as protein level. PBMCs were irradiated at various doses between 10 cGy to 2.0 Gy at a dose rate of 1.0 Gy/min. Total RNA was isolated for gene expression analysis at 0 hour and 4 hours respectively. cDNA was prepared and transcriptional pattern as studied using real time q-PCR where Taqman probes were used. Time point kinetics of transcriptional pattern of TRF1, TRF2, TIN2, TERF2, PTOP and POT1 was carried out at 0 min, 15 min, 30 min, 60 min, and 120 min for two different doses (1.0 Gy and 2.0 Gy). Dose response and time point kinetics of TRF2 was studied at similar doses using confocal microscopy. Our results revealed that at 2.0 Gy there was a two fold increase at the level of transcription

Solution structure of the twelfth cysteine-rich ligand-binding repeat in rat megalin

International Nuclear Information System (INIS)

Wolf, Christian A.; Dancea, Felician; Shi Meichen; Bade-Noskova, Veronika; Rueterjans, Heinz; Kerjaschki, Dontscho; Luecke, Christian

2007-01-01

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short β-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Energy Technology Data Exchange (ETDEWEB)

Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

2017-09-01

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis.

Science.gov (United States)

Wang, Lina; Tu, Zhaowei; Liu, Chao; Liu, Hongbin; Kaldis, Philipp; Chen, Zijiang; Li, Wei

2018-01-08

Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Trf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to-end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

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Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

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

Telomerase Repeated Amplification Protocol (TRAP).

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Mender, Ilgen; Shay, Jerry W

2015-11-20

Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al. , 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC - counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al. , 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

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de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

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Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2016-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2016-01-15

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

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Youn-Bok Lee

2013-12-01

Full Text Available The GGGGCC (G4C2 intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

DEFF Research Database (Denmark)

Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

2012-01-01

CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes.

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Steinhagen, Folkert; Zillinger, Thomas; Peukert, Konrad; Fox, Mario; Thudium, Marcus; Barchet, Winfried; Putensen, Christian; Klinman, Dennis; Latz, Eicke; Bode, Christian

2018-04-01

Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières Syndrome (AGS). The recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1-deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self-DNA. Collectively, these findings might lead to the development of new therapeutics against IFN-driven pathologies due to cGAS activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

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Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

2015-10-08

Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

Science.gov (United States)

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

Science.gov (United States)

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

Science.gov (United States)

Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

2017-11-01

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

Science.gov (United States)

Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

2015-12-02

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia

International Nuclear Information System (INIS)

Leitersdorf, E.; Hobbs, H.H.; Fourie, A.M.; Jacobs, M.; Van Der Westhuyzen, D.R.; Coetzee, G.A.

1988-01-01

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each ∼ 40 amino acids long. Previous studies showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125 I-labeled LDL but not 125 I-labeled IgG-C7. previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new Pst I restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate

Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes

DEFF Research Database (Denmark)

Holmsgaard, Peter Nikolai; Sørensen, Søren Johannes; Hansen, Lars H.

2013-01-01

The use of amplicon pyrosequencing makes it possible to produce thousands of sequences of the same gene at relatively low costs. Here we show that it is possible to simultaneously sequence the 16S rRNA gene, IncP-1 trfA gene and mercury reductase gene (merA) as a way for screening the diversity...

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

Science.gov (United States)

Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

2013-08-01

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

OpenAIRE

Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John?H.J.; Boulton, Simon?J.

2015-01-01

Summary The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to?prevent catastrophic t-loop processing by structure-specific nucleases. We show that ...

Human liver cell trafficking mutants: characterization and whole exome sequencing.

Directory of Open Access Journals (Sweden)

Fei Yuan

Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

Science.gov (United States)

Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

2015-06-01

One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

Repeated swim stress alters brain benzodiazepine receptors measured in vivo

International Nuclear Information System (INIS)

Weizman, R.; Weizman, A.; Kook, K.A.; Vocci, F.; Deutsch, S.I.; Paul, S.M.

1989-01-01

The effects of repeated swim stress on brain benzodiazepine receptors were examined in the mouse using both an in vivo and in vitro binding method. Specific in vivo binding of [ 3 H]Ro15-1788 to benzodiazepine receptors was decreased in the hippocampus, cerebral cortex, hypothalamus, midbrain and striatum after repeated swim stress (7 consecutive days of daily swim stress) when compared to nonstressed mice. In vivo benzodiazepine receptor binding was unaltered after repeated swim stress in the cerebellum and pons medulla. The stress-induced reduction in in vivo benzodiazepine receptor binding did not appear to be due to altered cerebral blood flow or to an alteration in benzodiazepine metabolism or biodistribution because there was no difference in [14C]iodoantipyrine distribution or whole brain concentrations of clonazepam after repeated swim stress. Saturation binding experiments revealed a change in both apparent maximal binding capacity and affinity after repeated swim stress. Moreover, a reduction in clonazepam's anticonvulsant potency was also observed after repeated swim stress [an increase in the ED50 dose for protection against pentylenetetrazol-induced seizures], although there was no difference in pentylenetetrazol-induced seizure threshold between the two groups. In contrast to the results obtained in vivo, no change in benzodiazepine receptor binding kinetics was observed using the in vitro binding method. These data suggest that environmental stress can alter the binding parameters of the benzodiazepine receptor and that the in vivo and in vitro binding methods can yield substantially different results

Repeated swim stress alters brain benzodiazepine receptors measured in vivo

Energy Technology Data Exchange (ETDEWEB)

Weizman, R.; Weizman, A.; Kook, K.A.; Vocci, F.; Deutsch, S.I.; Paul, S.M.

1989-06-01

The effects of repeated swim stress on brain benzodiazepine receptors were examined in the mouse using both an in vivo and in vitro binding method. Specific in vivo binding of (/sup 3/H)Ro15-1788 to benzodiazepine receptors was decreased in the hippocampus, cerebral cortex, hypothalamus, midbrain and striatum after repeated swim stress (7 consecutive days of daily swim stress) when compared to nonstressed mice. In vivo benzodiazepine receptor binding was unaltered after repeated swim stress in the cerebellum and pons medulla. The stress-induced reduction in in vivo benzodiazepine receptor binding did not appear to be due to altered cerebral blood flow or to an alteration in benzodiazepine metabolism or biodistribution because there was no difference in (14C)iodoantipyrine distribution or whole brain concentrations of clonazepam after repeated swim stress. Saturation binding experiments revealed a change in both apparent maximal binding capacity and affinity after repeated swim stress. Moreover, a reduction in clonazepam's anticonvulsant potency was also observed after repeated swim stress (an increase in the ED50 dose for protection against pentylenetetrazol-induced seizures), although there was no difference in pentylenetetrazol-induced seizure threshold between the two groups. In contrast to the results obtained in vivo, no change in benzodiazepine receptor binding kinetics was observed using the in vitro binding method. These data suggest that environmental stress can alter the binding parameters of the benzodiazepine receptor and that the in vivo and in vitro binding methods can yield substantially different results.

Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

Energy Technology Data Exchange (ETDEWEB)

Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith

2007-10-02

Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNA-binding proteins.

Science.gov (United States)

Zamiri, Bita; Reddy, Kaalak; Macgregor, Robert B; Pearson, Christopher E

2014-02-21

Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.

An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene

DEFF Research Database (Denmark)

Wade, M; Blake, M C; Jambou, R C

1995-01-01

consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes. Furthermore, the mutations...

Low-temperature-induced expression of rice ureidoglycolate amidohydrolase is mediated by a C-repeat/dehydration-responsive element that specifically interacts with rice C-repeat-binding factor 3

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Juan eLi

2015-11-01

Full Text Available Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH, which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT but not abscisic acid- (ABA regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (POsUAH. Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of POsUAH. Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

Science.gov (United States)

Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2015-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

Science.gov (United States)

Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M; Smith, Eric M; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

2017-12-01

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

Dissecting the telomere–inner nuclear membrane interface formed in meiosis

Energy Technology Data Exchange (ETDEWEB)

Pendlebury, Devon F.; Fujiwara, Yasuhiro; Tesmer, Valerie M.; Smith, Eric M.; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

2017-10-30

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere–INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1–TERB1 interface to reveal the structural basis for telomere–INM linkage. Disruption of this interface abrogates binding and compromises telomere–INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1–TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere–INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere–INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

Energy Technology Data Exchange (ETDEWEB)

Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M. (VA); (UCLA); (Vanderbilt); (UCSF)

2014-10-02

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Child Behavior Checklist (CBCL,Youth Self-Report (YSR and Teacher's Report Form(TRF: an overview of the development of the original and Brazilian versions Child Behavior Checklist (CBCL, Youth Self- Report (YSR y Teacher's Report Form (TRF: una visión general sobre el desarrollo de las versiones originales y brasileñas Child Behavior Checklist (CBCL, Youth Self-Report (YSR e Teacher's Report Form (TRF: uma visão geral sobre o desenvolvimento das versões originais e brasileiras

Directory of Open Access Journals (Sweden)

Isabel A. Bordin

2013-01-01

Full Text Available The Achenbach System of Empirically Based Assessment (ASEBA for school-age children includes three instruments for assessing emotional and/or behavioral problems: Child Behavior Checklist (CBCL, completed by parents, Youth Self-Report (YSR, completed by adolescents and Teacher's Report Form (TRF, completed by teachers. This review article gives detailed information on the development of these forms in the United States and Brazil, describing the main changes to the items, scales and score cut-off points in original versions between 1991 and 2001, as well as the process involved in the translation, back-translation and cultural adaptation of the original questionnaires to develop the current official Brazilian versions of the CBCL, YSR and TRF. The utility of these tools for research and clinical practice is highlighted, mentioning epidemiological studies and evaluation of interventions conducted in Brazil. Researchers' and clinicians' doubts regarding the correct use of the current official Brazilian versions are answered, giving examples of frequently asked questions relevant to the Brazilian context.El sistema de evaluación de base empírica de Achenbach para niños/adolescentes en edad escolar incluye tres instrumentos para evaluar problemas emocionales y/o de comportamiento: Child Behavior Checklist (CBCL [padres], Youth Self-Report (YSR [adolescentes] y Teacher's Report Form (TRF [profesores]. Este artículo de revisión proporciona información detallada sobre el desarrollo de estos instrumentos en los Estados Unidos y en Brasil, describiendo las principales alteraciones en ítems, escalas y puntos de corte en la puntuación, que se realizaron en las versiones originales de 1991 a 2001, y el proceso de traducción, retrotraducción y adaptación cultural de los cuestionarios originales, con el fin de desarrollar las actuales versiones brasileñas oficiales del CBCL, YSR y TRF. La utilidad de estos instrumentos en investigación y en la pr

The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

Science.gov (United States)

Fenyk, Stepan; Townsend, Philip D; Dixon, Christopher H; Spies, Gerhard B; de San Eustaquio Campillo, Alba; Slootweg, Erik J; Westerhof, Lotte B; Gawehns, Fleur K K; Knight, Marc R; Sharples, Gary J; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2015-10-09

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and Mapping of Simple Sequence Repeat Markers from Common Bean (Phaseolus vulgaris L. Bacterial Artificial Chromosome End Sequences for Genome Characterization and Genetic–Physical Map Integration

Directory of Open Access Journals (Sweden)

Juana M. Córdoba

2010-11-01

Full Text Available Microsatellite markers or simple sequence repeat (SSR loci are useful for diversity characterization and genetic–physical mapping. Different in silico microsatellite search methods have been developed for mining bacterial artificial chromosome (BAC end sequences for SSRs. The overall goal of this study was genome characterization based on SSRs in 89,017 BAC end sequences (BESs from the G19833 common bean ( L. library. Another objective was to identify new SSR taking into account three tandem motif identification programs (Automated Microsatellite Marker Development [AMMD], Tandem Repeats Finder [TRF], and SSRLocator [SSRL]. Among the microsatellite search engines, SSRL identified the highest number of SSRs; however, when primer design was attempted, the number dropped due to poor primer design regions. Automated Microsatellite Marker Development software identified many SSRs with valuable AT/TA or AG/TC motifs, while TRF found fewer SSRs and produced no primers. A subgroup of 323 AT-rich, di-, and trinucleotide SSRs were selected from the AMMD results and used in a parental survey with DOR364 and G19833, of which 75 could be mapped in the corresponding population; these represented 4052 BAC clones. Together with 92 previously mapped BES- and 114 non-BES-derived markers, a total of 280 SSRs were included in the polymerase chain reaction (PCR-based map, integrating a total of 8232 BAC clones in 162 contigs from the physical map.

The decorin sequence SYIRIADTNIT binds collagen type I

DEFF Research Database (Denmark)

Kalamajski, Sebastian; Aspberg, Anders; Oldberg, Ake

2007-01-01

Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site......-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro....... These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins....

Revisiting the TALE repeat.

Science.gov (United States)

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

Random mutagenesis of the nucleotide-binding domain of NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies gain-of-function mutations in the nucleotide-binding pocket

NARCIS (Netherlands)

Sueldo, D.J.; Shimels, M.Z.; Spiridon, L.N.; Caldararu, O.; Petrescu, A.J.; Joosten, M.H.A.J.; Tameling, W.I.L.

2015-01-01

•Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. •NRC1 (NB-LRR

Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione.

Directory of Open Access Journals (Sweden)

Dmitry Kaluzhny

Full Text Available Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethylamino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K(ass∼10⁶ M⁻¹ for human telomeric antiparallel quadruplex d(TTAGGG₄ and duplex d(TTAGGG₄∶d(CCCTAA₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na+ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K+ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

Science.gov (United States)

Kwon, Ilmin; Xiang, Siheng; Kato, Masato; Wu, Leeju; Theodoropoulos, Pano; Wang, Tao; Kim, Jiwoong; Yun, Jonghyun; Xie, Yang; McKnight, Steven L

2014-09-05

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death. Copyright © 2014, American Association for the Advancement of Science.

Expansion of protein domain repeats.

Directory of Open Access Journals (Sweden)

Asa K Björklund

2006-08-01

Full Text Available Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

Protein composition of catalytically active human telomerase from immortal cells

DEFF Research Database (Denmark)

Cohen, Scott B; Graham, Mark E; Lovrecz, George O

2007-01-01

Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on th...

Meta-analysis of the effect of overexpression of C-repeat/dehydration-responsive element binding family genes on temperature stress tolerance and related responses

Science.gov (United States)

C-repeat/dehydration-responsive element binding proteins are transcription factors that play a critical role in plant response to temperature stress. Over-expression of CBF/DREB genes has been demonstrated to enhance temperature stress tolerance. A series of physiological and biochemical modificat...

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

Science.gov (United States)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

RPA and POT1: friends or foes at telomeres?

Science.gov (United States)

Flynn, Rachel Litman; Chang, Sandy; Zou, Lee

2012-02-15

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.

Deciphering the Role of Alternative Non-Homologous End Joining (Alt-NHEJ) DNA Repair in Breast Cancer

Science.gov (United States)

2017-12-01

consecutive TTAGGG repeats. To detect rare reads containing fusion junctions, we exploited the novel sequence arrangement created by the ligation of the 39G...journal.pgen.1001005 (2010). 14 van Kregten, M. et al. T-DNA integration in plants results from polymerase-theta-mediated DNA repair. Nat Plants 2

StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

Science.gov (United States)

Xu, Yongtao; Zhou, Xu; Huang, Meilan

2015-01-01

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

Expression, purification, crystallization and preliminary X-ray diffraction studies of the human keratin 4-binding domain of serine-rich repeat protein 1 from Streptococcus agalactiae

International Nuclear Information System (INIS)

Sundaresan, Ramya; Samen, Ulrike; Ponnuraj, Karthe

2011-01-01

Expression, purification and crystallization of Srr-1-K4BD, a human keratin 4-binding domain of serine-rich repeat protein 1 from S. agalactiae, was carried out. Native crystals of Srr-1-K4BD diffracted to 3.8 Å resolution using synchrotron radiation. Serine-rich repeat protein 1 (Srr-1) is a surface protein from Streptococcus agalactiae. A 17 kDa region of this protein has been identified to bind to human keratin 4 (K4) and is termed the Srr-1 K4-binding domain (Srr-1-K4BD). Recombinant Srr-1-K4BD was overexpressed in Escherichia coli BL21 (DE3) cells. Native and selenomethionine-substituted proteins were prepared using Luria–Bertani (LB) and M9 minimal media, respectively. A two-step purification protocol was carried out to obtain a final homogenous sample of Srr-1-K4BD. Crystals of native Srr-1-K4BD were obtained using PEG 3350 as a precipitant. The crystals diffracted to 3.8 Å resolution using synchrotron radiation and belonged to space group P2 1 , with unit-cell parameters a = 47.56, b = 59.48, c = 94.71 Å, β = 93.95°

Ia-restricted B-B cell interaction. I. The MHC haplotype of bone marrow cells present during B cell ontogeny dictates the self-recognition specificity of B cells in the polyclonal B cell activation by a B cell differentiation factor, B151-TRF2

International Nuclear Information System (INIS)

Ono, S.; Takahama, Y.; Hamaoka, T.

1987-01-01

We have demonstrated that B cell recognition of Ia molecules is involved in polyclonal B cell differentiation by B151-TRF2. The present study was undertaken to examine the Ia recognition specificity of B151-TRF2-responsive B cells in fully major histocompatibility complex (MHC)-allogeneic P1----P2, semiallogeneic P1----(P1 x P2)F1, and double donor (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 radiation bone marrow chimeras. The B cells from both P1----P2 and P1----(P1 x P2)F1 chimeras could give rise to in vitro immunoglobulin M-producing cells upon stimulation with B151-TRF2 comparable in magnitude to that of normal P1 B cells, and their responses were inhibited by anti-I-AP1 but not by anti-I-AP2 monoclonal antibody even in the presence of mitomycin C-treated T cell-depleted P2 spleen cells as auxiliary cells. In contrast, the B151-TRF2 responses of P1 B cells isolated from both (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 double bone marrow chimeras became sensitive to the inhibition of not only anti-I-AP1 but also anti-I-AP2 monoclonal antibody only when the culture was conducted in the presence of P2 auxiliary cells, demonstrating that they adaptively differentiate to recognize as self-structures allogeneic as well as syngeneic Ia molecules. Moreover, the experiments utilizing B cells from H-2-congenic mice and B cell hybridoma clones as auxiliary cells revealed that B151-TRF2-responsive B cells recognize Ia molecules expressed on B cells. Taken together, these results demonstrate that B151-TRF2-responsive B cells recognize Ia molecules expressed by B cells as self-structures and that their self-recognition specificity is dictated by the MHC haplotype of bone marrow cells present during the B cell ontogeny but not by the MHC haplotype of a radiation-resistant host environment

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

Science.gov (United States)

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

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

KAUST Repository

Li, Lixin

2013-07-01

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

Repeated Vaccination of Cows with HIV Env gp140 during Subsequent Pregnancies Elicits and Sustains an Enduring Strong Env-Binding and Neutralising Antibody Response.

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Behnaz Heydarchi

Full Text Available An important feature of a potential vaccine against HIV is the production of broadly neutralising antibodies (BrNAbs capable of potentially blocking infectivity of a diverse array of HIV strains. BrNAbs naturally arise in some HIV infected individuals after several years of infection and their serum IgG can neutralise various HIV strains across different subtypes. We previously showed that vaccination of cows with HIV gp140 AD8 trimers resulted in a high titre of serum IgG against HIV envelope (Env that had strong BrNAb activity. These polyclonal BrNAbs concentrated into the colostrum during the late stage of pregnancy and can be harvested in vast quantities immediately after calving. In this study, we investigated the effect of prolonged HIV gp140 vaccination on bovine colostrum IgG HIV Env-binding and BrNAb activity over subsequent pregnancies. Repeated immunisation led to a maintained high titre of HIV Env specific IgG in the colostrum batches, but this did not increase through repeated cycles. Colostrum IgG from all batches also strongly competed with sCD4 binding to gp140 Env trimer and with human-derived monoclonal VRC01 and b12 BrNAbs that bind the CD4 binding site (CD4bs. Furthermore, competition neutralisation assays using RSC3 Env gp120 protein core and a derivative CD4bs mutant, RSC3 Δ371I/P363N, showed that CD4bs neutralising antibodies contribute to the neutralising activity of all batches of purified bovine colostrum IgG. This result indicates that the high IgG titre/avidity of anti-CD4bs antibodies with BrNAb activity was achieved during the first year of vaccination and was sustained throughout the years of repeated vaccinations in the cow tested. Although IgG of subsequent colostrum batches may have a higher avidity towards the CD4bs, the overall breadth in neutralisation was not enhanced. This implies that the boosting vaccinations over 4 years elicited a polyclonal antibody response that maintained the proportion of both

Interplay between I308 and Y310 residues in the third repeat of microtubule-binding domain is essential for tau filament formation.

Science.gov (United States)

Naruto, Keiko; Minoura, Katsuhiko; Okuda, Ryouhei; Taniguchi, Taizo; In, Yasuko; Ishida, Toshimasa; Tomoo, Koji

2010-10-08

Investigation of the mechanism of tau polymerization is indispensable for finding inhibitory conditions or identifying compounds preventing the formation of paired helical filament or oligomers. Tau contains a microtubule-binding domain consisting of three or four repeats in its C-terminal half. It has been considered that the key event in tau polymerization is the formation of a β-sheet structure arising from a short hexapeptide (306)VQIVYK(311) in the third repeat of tau. In this paper, we report for the first time that the C-H⋯π interaction between Ile308 and Tyr310 is the elemental structural scaffold essential for forming a dry "steric zipper" structure in tau amyloid fibrils. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Differential effects of acute and repeated electrically and chemically induced seizures on ( sup 3 H)Nimodipine and ( sup 125 I)omega-conotoxin GVIA binding in rat brain

Energy Technology Data Exchange (ETDEWEB)

Gleiter, C.H.; Cain, C.J.; Weiss, S.R.; Post, R.M.; Marangos, P.J. (National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD (USA))

1989-07-01

({sup 3}H)Nimodipine and high-affinity ({sup 125}I)omega-conotoxin GVIA (CgTX) binding were investigated in membranes from rat cerebral cortex, cerebellum, and hippocampus after electrically and chemically induced seizures. Animals were decapitated 30 min after a single electroconvulsive shock (ECS) or lidocaine-induced seizure and 24 h after the last of 10 once-daily ECS or six once-daily lidocaine-induced seizures. After a single ECS, ({sup 3}H)nimodipine and ({sup 125}I)CgTX binding sites decreased in cerebral cortex (by 10% and 17%, respectively). A downregulation of ({sup 3}H)nimodipine binding sites in hippocampus occurred after single and repeated lidocaine-induced seizures (by 24% and 11%, respectively), whereas ({sup 125}I)CgTX binding remained unaltered. An earlier report on changes in ({sup 3}H)nitrendipine binding after chronic ECS in cortex and hippocampus was not confirmed.

Repeated administration of D-amphetamine induces loss of [123I]FP-CIT binding to striatal dopamine transporters in rat brain: a validation study

International Nuclear Information System (INIS)

Booij, Jan; Bruin, Kora de; Gunning, W. Boudewijn

2006-01-01

In recent years, several PET and SPECT studies have shown loss of striatal dopamine transporter (DAT) binding in amphetamine (AMPH) users. However, the use of DAT SPECT tracers to detect AMPH-induced changes in DAT binding has not been validated. We therefore examined if repeated administration of D-AMPH or methamphetamine (METH) may induce loss of binding to striatal DATs in rats by using an experimental biodistribution study design and a SPECT tracer for the DAT ([ 123 I]FP-CIT). Methods: Groups of male rats (n=10 per group) were treated with D-AMPH (10 mg/kg body weight), METH (10 mg/kg body weight), or saline, twice a day for 5 consecutive days. Five days later, [ 123 I]FP-CIT was injected intravenously, and 2 h later, the rats were sacrificed and radioactivity was assayed. Results: In D-AMPH but not METH-treated rats, striatal [ 123 I]FP-CIT uptake was significantly lower (approximately 17%) than in the control group. Conclusion: These data show that [ 123 I]FP-CIT can be used to detect AMPH-induced changes in DAT binding and may validate the use of DAT radiotracers to study AMPH-induced changes in striatal DAT binding in vivo

Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

Energy Technology Data Exchange (ETDEWEB)

Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick; Campisi, Judith

2006-11-07

Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

Science.gov (United States)

Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

Science.gov (United States)

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Potential Role of the Last Half Repeat in TAL Effectors Revealed by a Molecular Simulation Study

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Hua Wan

2016-01-01

Full Text Available TAL effectors (TALEs contain a modular DNA-binding domain that is composed of tandem repeats. In all naturally occurring TALEs, the end of tandem repeats is invariantly a truncated half repeat. To investigate the potential role of the last half repeat in TALEs, we performed comparative molecular dynamics simulations for the crystal structure of DNA-bound TALE AvrBs3 lacking the last half repeat and its modeled structure having the last half repeat. The structural stability analysis indicates that the modeled system is more stable than the nonmodeled system. Based on the principle component analysis, it is found that the AvrBs3 increases its structural compactness in the presence of the last half repeat. The comparison of DNA groove parameters of the two systems implies that the last half repeat also causes the change of DNA major groove binding efficiency. The following calculation of hydrogen bond reveals that, by stabilizing the phosphate binding with DNA at the C-terminus, the last half repeat helps to adopt a compact conformation at the protein-DNA interface. It further mediates more contacts between TAL repeats and DNA nucleotide bases. Finally, we suggest that the last half repeat is required for the high-efficient recognition of DNA by TALE.

Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation.

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Guray Kuzu

2016-07-01

Full Text Available Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression.

Repeated administration of D-amphetamine induces loss of [{sup 123}I]FP-CIT binding to striatal dopamine transporters in rat brain: a validation study

Energy Technology Data Exchange (ETDEWEB)

Booij, Jan [Department of Nuclear Medicine, Academic Medical Center, 1105 AZ Amsterdam (Netherlands)]. E-mail: j.booij@amc.uva.nl; Bruin, Kora de [Department of Nuclear Medicine, Academic Medical Center, 1105 AZ Amsterdam (Netherlands); Gunning, W. Boudewijn [Department of Neurology, Epilepsy Centre Kempenhaeghe, 5590 AB Heeze (Netherlands)

2006-04-15

In recent years, several PET and SPECT studies have shown loss of striatal dopamine transporter (DAT) binding in amphetamine (AMPH) users. However, the use of DAT SPECT tracers to detect AMPH-induced changes in DAT binding has not been validated. We therefore examined if repeated administration of D-AMPH or methamphetamine (METH) may induce loss of binding to striatal DATs in rats by using an experimental biodistribution study design and a SPECT tracer for the DAT ([{sup 123}I]FP-CIT). Methods: Groups of male rats (n=10 per group) were treated with D-AMPH (10 mg/kg body weight), METH (10 mg/kg body weight), or saline, twice a day for 5 consecutive days. Five days later, [{sup 123}I]FP-CIT was injected intravenously, and 2 h later, the rats were sacrificed and radioactivity was assayed. Results: In D-AMPH but not METH-treated rats, striatal [{sup 123}I]FP-CIT uptake was significantly lower (approximately 17%) than in the control group. Conclusion: These data show that [{sup 123}I]FP-CIT can be used to detect AMPH-induced changes in DAT binding and may validate the use of DAT radiotracers to study AMPH-induced changes in striatal DAT binding in vivo.

Cloning of Ammopiptanthus mongolicus C-repeat-binding factor gene and its cold-induced tolerance in transgenic tobacco

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Lijiang Gu

2013-12-01

Full Text Available C-repeat-binding factors (CBFs are a type of important regulon in stress-related signal transduction pathways that control plant tolerance of abiotic stress. Ammopiptanthus mongolicus is the only evergreen broadleaf shrub in the northwest desert of China. The species shows strong resistance to environmental stress, especially to cold stress. An A. mongolicus CBF1 gene (AmCBF1 was cloned and transformed into tobacco. Expression of AmCBF1 could be detected in A. mongolicus shortly after exposure to low temperature of 4°C. Analysis on ratio of electrolytic leakage, soluble sugar content, free proline content, malondialdehyde (MDA content and peroxidase (POD activity before and after cold treatment (4°C for 24 h indicated AmCBF1 conferred higher cold tolerance to AmCBF1 transgenic tobacco compared with the wild type and empty vector transformed tobacco.

Time-Resolved Fluorescence of Water-Soluble Pyridinium Salt: Sensitive Detection of the Conformational Changes of Bovine Serum Albumin.

Science.gov (United States)

Li, Lei; Yi, Hua; Jia, Menghui; Chang, Mengfang; Zhou, Zhongneng; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Chen, Jinquan; Xu, Jianhua

2016-06-20

In this paper, we report a pyridinium salt "turn-on" fluorescent probe, 4-[2-(4-Dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (p-DASPMI), and applied its time-resolved fluorescence (TRF) to monitor the protein conformational changes. Both the fluorescence lifetime and quantum yield (QY) of p-DASPMI were increased about two orders of magnitude after binding to the protein bovine serum albumin (BSA). The free p-DASPMI in solution presents an ultrashort fluorescence lifetime (12.4 ps), thus it does not interfere the detection of bound p-DASPMI which has nanosecond fluorescence lifetime. Decay-associated spectra (DAS) show that p-DASPMI molecules bind to subdomains IIA and IIIA of BSA. The TRF decay profiles of p-DASPMI can be described by multi-exponential decay function ([Formula: see text]), and the obtained parameters, such as lifetimes ([Formula: see text]), fractional amplitudes ([Formula: see text]), and fractional intensities ([Formula: see text]), may be used to deduce the conformational changes of BSA. The pH and Cu 2+ induced conformational changes of BSA were investigated through the TRF of p-DASPMI. The results show that the p-DASPMI is a candidate fluorescent probe in studying the conformational changes of proteins through TRF spectroscopy and microscopy in the visible range. © The Author(s) 2016.

Karyological characterization and identification of four repetitive element groups (the 18S – 28S rRNA gene, telomeric sequences, microsatellite repeat motifs, Rex retroelements) of the Asian swamp eel (Monopterus albus)

Science.gov (United States)

Suntronpong, Aorarat; Thapana, Watcharaporn; Twilprawat, Panupon; Prakhongcheep, Ornjira; Somyong, Suthasinee; Muangmai, Narongrit; Surin Peyachoknagul; Srikulnath, Kornsorn

2017-01-01

Abstract Among teleost fishes, Asian swamp eel (Monopterus albus Zuiew, 1793) possesses the lowest chromosome number, 2n = 24. To characterize the chromosome constitution and investigate the genome organization of repetitive sequences in M. albus, karyotyping and chromosome mapping were performed with the 18S – 28S rRNA gene, telomeric repeats, microsatellite repeat motifs, and Rex retroelements. The 18S – 28S rRNA genes were observed to the pericentromeric region of chromosome 4 at the same position with large propidium iodide and C-positive bands, suggesting that the molecular structure of the pericentromeric regions of chromosome 4 has evolved in a concerted manner with amplification of the 18S – 28S rRNA genes. (TTAGGG)n sequences were found at the telomeric ends of all chromosomes. Eight of 19 microsatellite repeat motifs were dispersedly mapped on different chromosomes suggesting the independent amplification of microsatellite repeat motifs in M. albus. Monopterus albus Rex1 (MALRex1) was observed at interstitial sites of all chromosomes and in the pericentromeric regions of most chromosomes whereas MALRex3 was scattered and localized to all chromosomes and MALRex6 to several chromosomes. This suggests that these retroelements were independently amplified or lost in M. albus. Among MALRexs (MALRex1, MALRex3, and MALRex6), MALRex6 showed higher interspecific sequence divergences from other teleost species in comparison. This suggests that the divergence of Rex6 sequences of M. albus might have occurred a relatively long time ago. PMID:29093797

Treatment-related fluctuation in Guillain-Barre syndrome

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Thirunavukkarasu Thivakaran

2011-01-01

Full Text Available Guillain-Barre syndrome (GBS is usually a monophasic illness but relapses occur. A 55-year-old female with hypertension and vitiligo presented with acute inflammatory demyelinating polyradiculoneuropathy. She improved with immunoglobulin treatment started on day 6 of illness, but relapsed on day 14 warranting repeat immunoglobulin therapy. Thereafter recovery was complete. Her relapse was due to treatment-related fluctuation (TRF. TRF is improvement in the GBS disability scale of at least one grade after completion of immunotherapy followed by worsening of the disability scale of at least one grade within the first 2 months after disease onset. Recurrent GBS and chronic inflammatory demyelinating polyradiculoneuropathy were excluded. During the peak of the illness ANA titres were transiently high. The presence of other medical conditions, predominant proximal weakness and the absence of preceding diarrhea are predictors for TRF seen in this patient. Early treatment and evidence of ongoing immune activation have contributed toward TRF.

Calcium-dependent binding of Escherichia coli alpha-hemolysin to erythrocytes

International Nuclear Information System (INIS)

Boehm, D.F.

1989-01-01

Alpha hemolysin (AH), a protein secreted by certain strains of Escherichia coli, causes lysis of erythrocytes (RBCs) and is cytotoxic for other cells. The primary structure of AH contains an eight amino acid sequence tandemly repeated 13 times near the C-terminus. These repeated sequences are essential for hemolytic activity. AH also requires an unknown modification by an accessory protein, Hly C, for hemolytic activity. The role of calcium in the interaction of Ah with RBCs was investigated using recombinant strains which produced active and inactive forms of the toxin. Hemolytic activity was calcium-dependent. Osmotic protection experiments and immunoblots of SDS-PAGE separated proteins from washed, toxin-treated RBCs showed that the binding of active AH to RBCs was calcium-dependent. Binding of active AH to RBCs increased the calcium permeability of RBC membranes and resulted in changes in membrane protein profiles. The changes in membrane proteins did not cause the lysis of the cells. These results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells. 45 Ca-autoradiography of the recombinant hemolysins separated by SDS-PAGE and transferred to nitrocellulose showed that active AH bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences since a deletion hemolysin missing 11 of the 13 repeated sequences did not bind calcium. This deletion hemolysin was non-hemolytic and did not bind to RBC membranes. Hemolysin lacking the Hly C modification was also non-hemolytic and did not bind to RBC membranes. This unmodified AH contained the repeated sequences and bound calcium as efficiently as active AH

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

Science.gov (United States)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-07-05

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

International Nuclear Information System (INIS)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-01-01

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable

Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Bhattacharya, Monolekha; Das, Amit Kumar

2011-01-01

Highlights: ► The regulatory sequences recognized by TcrX have been identified. ► The regulatory region comprises of inverted repeats segregated by 30 bp region. ► The mode of binding of TcrX with regulatory sequence is unique. ► In silico TcrX–DNA docked model binds one of the inverted repeats. ► Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has not been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by ∼30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.

C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

Science.gov (United States)

Haeusler, Aaron R; Donnelly, Christopher J; Periz, Goran; Simko, Eric A J; Shaw, Patrick G; Kim, Min-Sik; Maragakis, Nicholas J; Troncoso, Juan C; Pandey, Akhilesh; Sattler, Rita; Rothstein, Jeffrey D; Wang, Jiou

2014-03-13

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

Alterations of telomerase activity and terminal restriction fragment in gastric cancer and its premalignant lesions.

Science.gov (United States)

Yang, S M; Fang, D C; Luo, Y H; Lu, R; Battle, P D; Liu, W W

2001-08-01

In order to explore the role of alterations of telomerase activity and terminal restriction fragment (TRF) length in the development and progression of gastric cancer. Telomerase activity was detected in 176 specimens of gastric mucosa obtained through an operation or endoscopical biopsy by using the telomeric repeat amplification protocol (TRAP) assay. Meanwhile, the mean length of TRF was measured with the use of a Southern blot in part of those samples. Telomerase activity was detected in 14 of 57 (24.6%) chronic atrophy gastritis patients, six of 18 (33.3%) intestinal metaplasia patients, three of eight (37.5%) dysplasia patients and 60 of 65 (92.3%) gastric cancer patients, respectively. Normal gastric mucosa revealed no telomerase activity. No association was found between telomerase activity and any clinicopathological parameters. The mean TRF length was decreased gradually with age in normal mucosa and in gastric cancer tissue. Regression analysis demonstrated that the reduction rate in these tissues was 41 +/- 12 base pairs/year. Among 35 gastric cancers, TRF length was shown to be shorter in 20 cases (57.1%), similar in 12 cases (34.3%) and elongated in three cases (7.6%), compared to the corresponding adjacent tissues. The mean TRF length tended to decrease as the mucosa underwent chronic atrophy gastritis, intestinal metaplasia, dysplasia and into gastric cancer. The mean TRF length in gastric cancer was not statistically correlated with clinicopathological parameters and telomerase activity. Our results suggest that telomerase is expressed during the early stage of gastric carcinogenesis, and that the clinical significance of TRF length appears to be limited in gastric cancer.

Human telomeric DNA: G-quadruplex, i-motif and Watson–Crick double helix

Science.gov (United States)

Phan, Anh Tuân; Mergny, Jean-Louis

2002-01-01

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson–Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C·C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding. PMID:12409451

Radioisotopic evaluation of portal circulation

International Nuclear Information System (INIS)

Maliska, C.; Rosenthal, D.

1986-01-01

The use of a radio-tracer of portal circulation through the intestine, should prevent cruel punctures in the portal-vein or spleen as it is usually the case with traditional methods in the study of portal-system. The absorption of I-131 and Tc-99m, previously cheked in rabbits presented similar results in dogs. The time of circulation between terminal large-intestine and the liver (t-RF) was determined by external counting at hepatic level by recording radioactivity variation-time. In healthy animals the t-RF was from 20to 60 seconds, with average time of 42 seconds. In 2 animals with partial binding of portal-vein the t-RF went up to 110 and 120 seconds. (Author) [pt

Gentamicin binds to the megalin receptor as a competitive inhibitor using the common ligand binding motif of complement type repeats

DEFF Research Database (Denmark)

Dagil, Robert; O'Shea, Charlotte; Nykjær, Anders

2013-01-01

megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described...... to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists....

Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei

International Nuclear Information System (INIS)

Sabourin, P.J.; Sun, J.D.; MacGregor, J.T.; Wehr, C.M.; Birnbaum, L.S.; Lucier, G.; Henderson, R.F.

1990-01-01

Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344/N rats and B6C3F1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased

Alanine repeats influence protein localization in splicing speckles and paraspeckles.

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Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

2014-12-16

Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

Science.gov (United States)

Zhang, Xiaorong; Yarman, Aysu; Erdossy, Júlia; Katz, Sagie; Zebger, Ingo; Jetzschmann, Katharina J; Altintas, Zeynep; Wollenberger, Ulla; Gyurcsányi, Róbert E; Scheller, Frieder W

2018-05-15

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered. Copyright © 2018 Elsevier B.V. All rights reserved.

High expression of TRF2, SOX10, and CD10 in circulating tumor microemboli detected in metastatic melanoma patients. A potential impact for the assessment of disease aggressiveness.

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Long, Elodie; Ilie, Marius; Bence, Coraline; Butori, Catherine; Selva, Eric; Lalvée, Salomé; Bonnetaud, Christelle; Poissonnet, Gilles; Lacour, Jean-Philippe; Bahadoran, Philippe; Brest, Patrick; Gilson, Eric; Ballotti, Robert; Hofman, Véronique; Hofman, Paul

2016-06-01

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method. We characterized the phenotype of CTCs using anti-PS100, anti-SOX10, anti-CD10, and anti-TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow-up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Feature binding in visual short term memory: A General Recognition Theory analysis.

Science.gov (United States)

Fitousi, Daniel

2017-05-23

Creating and maintaining accurate bindings of elementary features (e.g., color and shape) in visual short-term memory (VSTM) is fundamental for veridical perception. How are low-level features bound in memory? The present work harnessed a multivariate model of perception - the General Recognition Theory (GRT) - to unravel the internal representations underlying feature binding in VSTM. On each trial, preview and target colored shapes were presented in succession, appearing in either repeated or altered spatial locations. Participants gave two same/different responses: one with respect to color and one with respect to shape. Converging GRT analyses on the accuracy confusion matrices provided substantial evidence for binding in the form of violations of perceptual independence at the level of the individual stimulus, such that positive correlations were obtained when both features repeated or alternated together, while negative correlations were obtained when one feature repeated and the other alternated. This "cloverleaf" GRT pattern of binding was similar whether the spatial location of the preview and target repeated or altered. The current results are consistent with: (a) the discrete memory "slots" model of VSTM, and (b) the notion that spatial location is not necessary for the formation of "object files." The GRT approach presented here offers a viable quantitative model for testing various questions regarding feature binding in VSTM.

Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites.

Science.gov (United States)

Friedrich, Nikolas; Santos, Joana M; Liu, Yan; Palma, Angelina S; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

2010-01-15

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for alpha2-3- over alpha2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to alpha2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6'sulfo-sialyl Lewis(x) might have implications for tissue tropism.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

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Shanshan Huang

2015-10-01

Full Text Available In polyglutamine (polyQ diseases, large polyQ repeats cause juvenile cases with different symptoms than those of adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knockin mouse models of spinal cerebellar ataxia-17 (SCA17, we found that a large polyQ (105 glutamines in the TATA-box-binding protein (TBP preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

Science.gov (United States)

Huang, Shanshan; Yang, Su; Guo, Jifeng; Yan, Sen; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2015-01-01

SUMMARY In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knock-in mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases. PMID:26387956

Characterization of a second ligand binding site of the insulin receptor

International Nuclear Information System (INIS)

Hao Caili; Whittaker, Linda; Whittaker, Jonathan

2006-01-01

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

International Nuclear Information System (INIS)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

2005-01-01

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

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

KAUST Repository

Li, Lixin; Atef, Ahmed; Piatek, Agnieszka Anna; Ali, Zahir; Piatek, Marek J.; Aouida, Mustapha; Sharakuu, Altanbadralt; Mahjoub, Ali; Wang, Guangchao; Khan, Mohammad Suhail; Fedoroff, Nina V.; Zhu, Jiankang; Mahfouz, Magdy M.

2013-01-01

, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA

Partial characterization of GTP-binding proteins in Neurospora

International Nuclear Information System (INIS)

Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

1987-01-01

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

Sequestration of DROSHA and DGCR8 by Expanded CGG RNA Repeats Alters MicroRNA Processing in Fragile X-Associated Tremor/Ataxia Syndrome

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Chantal Sellier

2013-03-01

Full Text Available Fragile X-associated tremor/ataxia syndrome (FXTAS is an inherited neurodegenerative disorder caused by the expansion of 55–200 CGG repeats in the 5′ UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.

Epitopes of MUC1 Tandem Repeats in Cancer as Revealed by Antibody Crystallography: Toward Glycopeptide Signature-Guided Therapy

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Dapeng Zhou

2018-05-01

Full Text Available Abnormally O-glycosylated MUC1 tandem repeat glycopeptide epitopes expressed by multiple types of cancer have long been attractive targets for therapy in the race against genetic mutations of tumor cells. Glycopeptide signature-guided therapy might be a more promising avenue than mutation signature-guided therapy. Three O-glycosylated peptide motifs, PDTR, GSTA, and GVTS, exist in a tandem repeat HGVTSAPDTRPAPGSTAPPA, containing five O-glycosylation sites. The exact peptide and sugar residues involved in antibody binding are poorly defined. Co-crystal structures of glycopeptides and respective monoclonal antibodies are very few. Here we review 3 groups of monoclonal antibodies: antibodies which only bind to peptide portion, antibodies which only bind to sugar portion, and antibodies which bind to both peptide and sugar portions. The antigenicity of peptide and sugar portions of glyco-MUC1 tandem repeat were analyzed according to available biochemical and structural data, especially the GSTA and GVTS motifs independent from the most studied PDTR. Tn is focused as a peptide-modifying residue in vaccine design, to induce glycopeptide-binding antibodies with cross reactivity to Tn-related tumor glycans, but not glycans of healthy cells. The unique requirement for the designs of antibody in antibody-drug conjugate, bi-specific antibodies, and chimeric antigen receptors are also discussed.

A comparative study on pathological features of transgenic rat lines expressing either three or four repeat misfolded tau.

Science.gov (United States)

Valachova, Bernadeta; Brezovakova, Veronika; Bugos, Ondrej; Jadhav, Santosh; Smolek, Tomas; Novak, Petr; Zilka, Norbert

2018-08-01

Human tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by distinct clinical features, typical histopathological structures, and defined ratio(s) of three-repeat and four-repeat tau isoforms within pathological aggregates. How the optional microtubule-binding repeat of tau influences this differentiation of pathologies is understudied. We have previously generated and characterized transgenic rodent models expressing human truncated tau aa151-391 with either three (SHR24) or four microtubule-binding repeats (SHR72). Here, we compare the behavioral and neuropathological hallmarks of these two transgenic lines using a battery of tests for sensorimotor, cognitive, and neurological functions over the age range of 3.5-15 months. Progression of sensorimotor and neurological deficits was similar in both transgenic lines; however, the lifespan of transgenic line SHR72 expressing truncated four-repeat tau was markedly shorter than SHR24. Moreover, the expression of three or four-repeat tau induced distinct neurofibrillary pathology in these lines. Transgenic lines displayed different distribution of tau pathology and different type of neurofibrillary tangles. Our results suggest that three- and four-repeat isoforms of tau may display different modes of action in the diseased brain. © 2018 Wiley Periodicals, Inc.

Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats.

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Jing Zhao

Full Text Available The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1β. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1β production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1β, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.

Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells

International Nuclear Information System (INIS)

Denner, Joachim; Specke, Volker; Thiesen, Ulla; Karlas, Alexander; Kurth, Reinhard

2003-01-01

Human-tropic porcine endogenous retroviruses (PERV) such as PERV-A and PERV-B can infect human cells and are therefore a potential risk to recipients of xenotransplants. A similar risk is posed by recombinant viruses containing the receptor-binding site of PERV-A and large parts of the genome of the ecotropic PERV-C including its long terminal repeat (LTR). We describe here the unique organization of the PERV-C LTR and its changes during serial passage of recombinant virus in human cells. An increase in virus titer correlated with an increase in LTR length, caused by multiplication of 37-bp repeats containing nuclear factor Y binding sites. Luciferase dual reporter assays revealed a correlation between the number of repeats and the extent of expression. No alterations have been observed in the receptor-binding site, indicating that the increased titer is due to the changes in the LTR. These data indicate that recombinant PERVs generated during infection of human cells can adapt and subsequently replicate with greater efficiency

Natively Unfolded FG Repeats Stabilize the Structure of the Nuclear Pore Complex.

Science.gov (United States)

Onischenko, Evgeny; Tang, Jeffrey H; Andersen, Kasper R; Knockenhauer, Kevin E; Vallotton, Pascal; Derrer, Carina P; Kralt, Annemarie; Mugler, Christopher F; Chan, Leon Y; Schwartz, Thomas U; Weis, Karsten

2017-11-02

Nuclear pore complexes (NPCs) are ∼100 MDa transport channels assembled from multiple copies of ∼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

Science.gov (United States)

Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

2011-09-02

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

Science.gov (United States)

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

Non-immune binding of human IgG to M-related proteins confers resistance to phagocytosis of group A streptococci in blood.

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Harry S Courtney

Full Text Available The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp are group A streptococcal (GAS receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

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Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

2013-09-03

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

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Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

2013-01-01

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

Genome-wide identification and tissue-specific expression analysis of nucleotide binding site-leucine rich repeat gene family in Cicer arietinum (kabuli chickpea).

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Sharma, Ranu; Rawat, Vimal; Suresh, C G

2017-12-01

The nucleotide binding site-leucine rich repeat (NBS-LRR) proteins play an important role in the defense mechanisms against pathogens. Using bioinformatics approach, we identified and annotated 104 NBS-LRR genes in chickpea. Phylogenetic analysis points to their diversification into two families namely TIR-NBS-LRR and non-TIR-NBS-LRR. Gene architecture revealed intron gain/loss events in this resistance gene family during their independent evolution into two families. Comparative genomics analysis elucidated its evolutionary relationship with other fabaceae species. Around 50% NBS-LRRs reside in macro-syntenic blocks underlining positional conservation along with sequence conservation of NBS-LRR genes in chickpea. Transcriptome sequencing data provided evidence for their transcription and tissue-specific expression. Four cis -regulatory elements namely WBOX, DRE, CBF, and GCC boxes, that commonly occur in resistance genes, were present in the promoter regions of these genes. Further, the findings will provide a strong background to use candidate disease resistance NBS-encoding genes and identify their specific roles in chickpea.

MNS16A tandem repeat minisatellite of human telomerase gene: functional studies in colorectal, lung and prostate cancer.

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Hofer, Philipp; Zöchmeister, Cornelia; Behm, Christian; Brezina, Stefanie; Baierl, Andreas; Doriguzzi, Angelina; Vanas, Vanita; Holzmann, Klaus; Sutterlüty-Fall, Hedwig; Gsur, Andrea

2017-04-25

MNS16A, a functional polymorphic tandem repeat minisatellite, is located in the promoter region of an antisense transcript of the human telomerase reverse transcriptase gene. MNS16A promoter activity depends on the variable number of tandem repeats (VNTR) presenting varying numbers of transcription factor binding sites for GATA binding protein 1. Although MNS16A has been investigated in multiple cancer epidemiology studies with incongruent findings, functional data of only two VNTRs (VNTR-243 and VNTR-302) were available thus far, linking the shorter VNTR to higher promoter activity.For the first time, we investigated promoter activity of all six VNTRs of MNS16A in cell lines of colorectal, lung and prostate cancer using Luciferase reporter assay. In all investigated cell lines shorter VNTRs showed higher promoter activity. While this anticipated indirect linear relationship was affirmed for colorectal cancer SW480 (P = 0.006), a piecewise linear regression model provided significantly better model fit in lung cancer A-427 (P = 6.9 × 10-9) and prostate cancer LNCaP (P = 0.039). In silico search for transcription factor binding sites in MNS16A core repeat element suggested a higher degree of complexity involving X-box binding protein 1, general transcription factor II-I, and glucocorticoid receptor alpha in addition to GATA binding protein 1.Further functional studies in additional cancers are requested to extend our knowledge of MNS16A functionality uncovering potential cancer type-specific differences. Risk alleles may vary in different malignancies and their determination in vitro could be relevant for interpretation of genotype data.

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose.

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Ting-Ying Jiang

Full Text Available The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21 members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

Sequential binding of calcium ions to the B-repeat domain of SdrD from Staphylococcus aureus.

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Roman, Andrei Yu; Devred, François; Lobatchov, Vladimir M; Makarov, Alexander A; Peyrot, Vincent; Kubatiev, Aslan A; Tsvetkov, Philipp O

2016-02-01

Biofilms of live bacteria forming on medical devices and implants contribute significantly to bacterial blood dissemination and to the spread of nosocomial infections. Cell surface SdrD protein plays a key role in the attachment of Staphylococcus aureus to the extracellular matrix (ECM) and in the formation of biofilm. SdrD binds calcium ions using its B1-B5 region bearing EF-hand Ca-binding sites, leading to conformational changes in the structure of SdrD. This alters the distance between the bacterial surface and the ECM-interacting domain of SdrD in a spring-like fashion, participating in bacterial attachment. In this study we investigated calcium binding to EF-hand sites of SdrD using isothermal titration calorimetry and determined the impact of this process on SdrD's thermodynamic stability. This allowed us to propose a model of B1-B5 reorganization upon binding of calcium and to get new insight into the molecular mechanism of SdrD's action.

Detection, characterization and evolution of internal repeats in Chitinases of known 3-D structure.

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Manigandan Sivaji

Full Text Available Chitinase proteins have evolved and diversified almost in all organisms ranging from prokaryotes to eukaryotes. During evolution, internal repeats may appear in amino acid sequences of proteins which alter the structural and functional features. Here we deciphered the internal repeats from Chitinase and characterized the structural similarities between them. Out of 24 diverse Chitinase sequences selected, six sequences (2CJL, 2DSK, 2XVP, 2Z37, 3EBV and 3HBE did not contain any internal repeats of amino acid sequences. Ten sequences contained repeats of length <50, and the remaining 8 sequences contained repeat length between 50 and 100 residues. Two Chitinase sequences, 1ITX and 3SIM, were found to be structurally similar when analyzed using secondary structure of Chitinase from secondary and 3-Dimensional structure database of Protein Data Bank. Internal repeats of 3N17 and 1O6I were also involved in the ligand-binding site of those Chitinase proteins, respectively. Our analyses enhance our understanding towards the identification of structural characteristics of internal repeats in Chitinase proteins.

Genome-wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

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Eunyoung Seo

2016-08-01

Full Text Available Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR immune receptors are known play critical roles in effector-triggered immunity (ETI plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analyses and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analyses of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

Viral delivery of C9orf72 hexanucleotide repeat expansions in mice leads to repeat-length-dependent neuropathology and behavioural deficits

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Saul Herranz-Martin

2017-07-01

Full Text Available Intronic GGGGCC repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Two major pathologies stemming from the hexanucleotide RNA expansions (HREs have been identified in postmortem tissue: intracellular RNA foci and repeat-associated non-ATG dependent (RAN dipeptides, although it is unclear how these and other hallmarks of disease contribute to the pathophysiology of neuronal injury. Here, we describe two novel lines of mice that overexpress either 10 pure or 102 interrupted GGGGCC repeats mediated by adeno-associated virus (AAV and recapitulate the relevant human pathology and disease-related behavioural phenotypes. Similar levels of intracellular RNA foci developed in both lines of mice, but only mice expressing 102 repeats generated C9orf72 RAN pathology, neuromuscular junction (NMJ abnormalities, dispersal of the hippocampal CA1, enhanced apoptosis, and deficits in gait and cognition. Neither line of mice, however, showed extensive TAR DNA-binding protein 43 (TDP-43 pathology or neurodegeneration. Our data suggest that RNA foci pathology is not a good predictor of C9orf72 RAN dipeptide formation, and that RAN dipeptides and NMJ dysfunction are drivers of C9orf72 disease pathogenesis. These AAV-mediated models of C9orf72-associated ALS/FTD will be useful tools for studying disease pathophysiology and developing new therapeutic approaches.

Photoaffinity labelling of high affinity dopamine binding proteins

International Nuclear Information System (INIS)

Ross, G.M.; McCarry, B.E.; Mishra, R.K.

1986-01-01

A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-]N'-4-azidobenzamidol]-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using ( 3 H)-SCH 23390 (a D 1 specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked ( 3 H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of ( 3 H)-SCH 23390 binding. Compounds which compete for D 1 receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D 1 (adenylate cyclase linked) dopamine receptor

The effect of gamma-enhancing binaural beats on the control of feature bindings.

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Colzato, Lorenza S; Steenbergen, Laura; Sellaro, Roberta

2017-07-01

Binaural beats represent the auditory experience of an oscillating sound that occurs when two sounds with neighboring frequencies are presented to one's left and right ear separately. Binaural beats have been shown to impact information processing via their putative role in increasing neural synchronization. Recent studies of feature-repetition effects demonstrated interactions between perceptual features and action-related features: repeating only some, but not all features of a perception-action episode hinders performance. These partial-repetition (or binding) costs point to the existence of temporary episodic bindings (event files) that are automatically retrieved by repeating at least one of their features. Given that neural synchronization in the gamma band has been associated with visual feature bindings, we investigated whether the impact of binaural beats extends to the top-down control of feature bindings. Healthy adults listened to gamma-frequency (40 Hz) binaural beats or to a constant tone of 340 Hz (control condition) for ten minutes before and during a feature-repetition task. While the size of visuomotor binding costs (indicating the binding of visual and action features) was unaffected by the binaural beats, the size of visual feature binding costs (which refer to the binding between the two visual features) was considerably smaller during gamma-frequency binaural beats exposure than during the control condition. Our results suggest that binaural beats enhance selectivity in updating episodic memory traces and further strengthen the hypothesis that neural activity in the gamma band is critically associated with the control of feature binding.

HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment

NARCIS (Netherlands)

Kappei, D.; Butter, F.; Benda, C.; Scheibe, M.; Draskovic, Irena; Stevense, M.; Novo, C.L.; Basquin, C.; Araki, M.; Araki, K.; Krastev, D.B.; Kittler, R.; Jessberger, R.; Londono-Vallejo, J.A.; Mann, M.; Buchholz, F.

2013-01-01

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the

Contribution of the LIM domain and nebulin-repeats to the interaction of Lasp-2 with actin filaments and focal adhesions.

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Hiroyuki Nakagawa

Full Text Available Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1 retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

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Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R

2015-01-01

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

Directory of Open Access Journals (Sweden)

Cristina eFenollar Ferrer

2015-09-01

Full Text Available Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to either the outside or inside of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (asymmetry of these systems has been successfully used as a bioinformatic tool, called repeat-swap modeling to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that

Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

Energy Technology Data Exchange (ETDEWEB)

Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

2012-05-22

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

Effects of intermittent fasting on glucose and lipid metabolism.

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Antoni, Rona; Johnston, Kelly L; Collins, Adam L; Robertson, M Denise

2017-08-01

Two intermittent fasting variants, intermittent energy restriction (IER) and time-restricted feeding (TRF), have received considerable interest as strategies for weight-management and/or improving metabolic health. With these strategies, the pattern of energy restriction and/or timing of food intake are altered so that individuals undergo frequently repeated periods of fasting. This review provides a commentary on the rodent and human literature, specifically focusing on the effects of IER and TRF on glucose and lipid metabolism. For IER, there is a growing evidence demonstrating its benefits on glucose and lipid homeostasis in the short-to-medium term; however, more long-term safety studies are required. Whilst the metabolic benefits of TRF appear quite profound in rodents, findings from the few human studies have been mixed. There is some suggestion that the metabolic changes elicited by these approaches can occur in the absence of energy restriction, and in the context of IER, may be distinct from those observed following similar weight-loss achieved via modest continuous energy restriction. Mechanistically, the frequently repeated prolonged fasting intervals may favour preferential reduction of ectopic fat, beneficially modulate aspects of adipose tissue physiology/morphology, and may also impinge on circadian clock regulation. However, mechanistic evidence is largely limited to findings from rodent studies, thus necessitating focused human studies, which also incorporate more dynamic assessments of glucose and lipid metabolism. Ultimately, much remains to be learned about intermittent fasting (in its various forms); however, the findings to date serve to highlight promising avenues for future research.

Measurement of Telomere Length in Colorectal Cancers for Improved Molecular Diagnosis

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Eric Le Balc’h

2017-08-01

Full Text Available All tumors have in common to reactivate a telomere maintenance mechanism to allow for unlimited proliferation. On the other hand, genetic instability found in some tumors can result from the loss of telomeres. Here, we measured telomere length in colorectal cancers (CRCs using TRF (Telomere Restriction Fragment analysis. Telomeric DNA content was also quantified as the ratio of total telomeric (TTAGGG sequences over that of the invariable Alu sequences. In most of the 125 CRCs analyzed, there was a significant diminution in telomere length compared with that in control healthy tissue. Only 34 tumors exhibited no telomere erosion and, in some cases, a slight telomere lengthening. Telomere length did not correlate with age, gender, tumor stage, tumor localization or stage of tumor differentiation. In addition, while telomere length did not correlate with the presence of a mutation in BRAF (V-raf murine sarcoma viral oncogene homolog B, PIK3CA (phosphatidylinositol 3-kinase catalytic subunit, or MSI status, it was significantly associated with the occurrence of a mutation in KRAS. Interestingly, we found that the shorter the telomeres in healthy tissue of a patient, the larger an increase in telomere length in the tumor. Our study points to the existence of two types of CRCs based on telomere length and reveals that telomere length in healthy tissue might influence telomere maintenance mechanisms in the tumor.

Repeated cocaine administration results in supersensitivity of striatal D-2 dopamine autoreceptors to pergolide

International Nuclear Information System (INIS)

Dwoskin, L.P.; Peris, J.; Yasuda, R.P.; Philpott, K.; Zahniser, N.R.

1988-01-01

Groups of rats administered cocaine-HCl (10 mg/kg, i.p.) or saline either acutely or once daily for 8 or 14 days were killed 24 hrs after the last dose. In striatal slices prelabelled with [ 3 H]DA, modulation of [ 3 H]-overflow by pergolide was used to measure D-2 autoreceptor activity. Compared to the contemporaneous control group pergolide produced a greater inhibition only in striatal slices from rats treated repeatedly with cocaine. In radioligand binding studies using striatal membranes from control rats, pergolide had a 500-fold greater affinity for the D-2, as opposed to the D-1, dopamine (DA) receptor subtype. These results indicate that repeated treatment with cocaine produces supersensitive striatal D-2 release-modulating autoreceptors consistent with a compensatory change to diminish the effect of elevated synaptic concentrations of DA produced by cocaine. In contrast, supersensitivity of D-2 receptors was not detected in [ 3 H]spiperone binding assays. 31 references, 2 figures, 1 table

A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids

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Yu-Chen Chuang

2018-06-01

Full Text Available Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

Complexities in human herpesvirus-6A and -6B binding to host cells

DEFF Research Database (Denmark)

Pedersen, Simon Metz; Höllsberg, Per

2006-01-01

Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...

Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

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Finstad Samantha L

2004-09-01

Full Text Available Abstract Background Feline leukemia virus (FeLV induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation. Results Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication. Conclusion Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in

In silico engineering and optimization of Transcription Activator-Like Effectors and their derivatives for improved DNA binding predictions.

KAUST Repository

Piatek, Marek J.

2015-12-01

Transcription Activator-Like Effectors (TALEs) can be used as adaptable DNAbinding modules to create site-specific chimeric nucleases or synthetic transcriptional regulators. The central repeat domain mediates specific DNA binding via hypervariable repeat di-residues (RVDs). This DNA-Binding Domain can be engineered to bind preferentially to any user-selected DNA sequence if engineered appropriately. Therefore, TALEs and their derivatives have become indispensable molecular tools in site-specific manipulation of genes and genomes. This thesis revolves around two problems: in silico design and improved binding site prediction of TALEs. In the first part, a study is shown where TALEs are successfully designed in silico and validated in laboratory to yield the anticipated effects on selected genes. Software is developed to accompany the process of designing and prediction of binding sites. I expanded the functionality of the software to be used as a more generic set of tools for the design, target and offtarget searching. Part two contributes a method and associated toolkit developed to allow users to design in silico optimized synthetic TALEs with user-defined specificities for various experimental purposes. This method is based on a mutual relationship of three consecutive tandem repeats in the DNA-binding domain. This approach revealed positional and compositional bias behind the binding of TALEs to DNA. In conclusion, I developed methods, approaches, and software to enhance the functionality of synthetic TALEs, which should improve understanding of TALEs biology and will further advance genome-engineering applications in various organisms and cell types.

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

Science.gov (United States)

Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

2008-01-01

Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Structure of filamin A immunoglobulin-like repeat 10 from Homo sapiens

International Nuclear Information System (INIS)

Page, Richard C.; Clark, Jeffrey G.; Misra, Saurav

2011-01-01

The structure of immunoglobulin-like repeat 10 from human filamin A solved at 2.44 Å resolution suggests the potential effects of mutations correlated with otopalatodigital syndrome spectrum disorders. Filamin A (FlnA) plays a critical role in cytoskeletal organization, cell motility and cellular signaling. FlnA utilizes different binding sites on a series of 24 immunoglobulin-like domains (Ig repeats) to interact with diverse cytosolic proteins and with cytoplasmic portions of membrane proteins. Mutations in a specific domain, Ig10 (FlnA-Ig10), are correlated with two severe forms of the otopalatodigital syndrome spectrum disorders Melnick–Needles syndrome and frontometaphyseal dysplasia. The crystal structure of FlnA-Ig10 determined at 2.44 Å resolution provides insight into the perturbations caused by these mutations

TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

Science.gov (United States)

Pereira, L A; van der Knaap, J A; van den Boom, V; van den Heuvel, F A; Timmers, H T

2001-11-01

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

Contribution to the study of TRH (thyrotropin-releasing hormone) conformation using circular dichroism. Physico-chemical studies, radioactive labelling and biological applications

International Nuclear Information System (INIS)

Pradelles, Philippe.

1977-01-01

In an attempt to reach a better understanding at the molecular level of phenomena connected with the action of TRF the conformation and radioactive labelling of this hormone were investigated. The specific detection of a hormone at its action site is only possible if labelled substances of very high specific activity are used. TRF was tritium labelled by three methods: direct catalytic exchange; catalytic dehalogenation of mono- and di-iodo TRF; catalytic denitrogenation of mono-azo-TRF. Whatever the method used the tritiated TRF has a very high specific activity and keeps all its biological properties. Biological activity measurements carried out on labelled TRF, in vivo in rats and in vitro on a TRF-sensitive prolactine cell clone, are described. TRF tritiated by the above methods is shown to have the same biological activity as standard TRF. Some results are given concerning the application of labelled TRF to research on the hormone action mechanism. The tritiated TRF distribution kinetics were examined in vivo and in vitro. The kinetics of hormone fixation on the antehypophysary tissue match those of in vivo release of the plasma thyreotropic hormone, confirming the relationships between the hormone fixation on its target tissue and its biological effect. Finally an outline is given of work on the interaction of tritiated TRF with prolactine cell receptors and on the penetration of intact tritiated TRF into these cells. In addition the radioimmunological analysis of TRF was developed by the use of 125 I-mono-iodo-TRF at high specific activity (above 2000 Ci/mmole) [fr

Low-Normal FMR1 CGG Repeat Length: Phenotypic Associations

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Marsha eMailick

2014-09-01

Full Text Available This population-based study investigates genotype-phenotype correlations of low-normal CGG repeats in the fragile X mental retardation 1 (FMR1 gene. FMR1 plays an important role in brain development and function, and encodes FMRP (fragile X mental retardation protein, an RNA-binding protein that regulates protein synthesis impacting activity-dependent synaptic development and plasticity. Most past research has focused on CGG premutation expansions (41 to 200 CGG repeats and on fragile X syndrome (200+ CGG repeats, with considerably less attention on the other end of the spectrum of CGG repeats. Using existing data, older adults with 23 or fewer CGG repeats (2 SDs below the mean were compared with age-peers who have normal numbers of CGGs (24-40 with respect to cognition, mental health, cancer, and having children with disabilities. Men (n = 341 with an allele in the low-normal range and women (n = 46 with two low-normal alleles had significantly more difficulty with their memory and ability to solve day to day problems. Women with both FMR1 alleles in the low-normal category had significantly elevated odds of feeling that they need to drink more to get the same effect as in the past. These women also had two and one-half times the odds of having had breast cancer and four times the odds of uterine cancer. Men and women with low-normal CGGs had higher odds of having a child with a disability, either a developmental disability or a mental health condition. These findings are in line with the hypothesis that there is a need for tight neuronal homeostatic control mechanisms for optimal cognitive and behavioral functioning, and more generally that low numbers as well as high numbers of CGG repeats may be problematic for health.

125I-iomazenil binding shows stress- and/or diazepam-induced reductions in mouse brain. Supporting data for 123I-iomazenil SPECT study of anxiety disorders

International Nuclear Information System (INIS)

Takahashi, Makoto; Odano, Ikuo; Fujita, Shozo; Ohkubo, Masaki.

1997-01-01

Effects of repeated swim stress on the binding of 125 I-iomazenil were examined in the brains of diazepam-treated and non-treated mice. The mice were orally administered diazepam or vehicle (0.5% ethylene glycol) and subjected to daily swim stress (at 20degC for 10 min) for seven consecutive days. The distribution and the amount of 125 I-iomazenil binding were analyzed autoradiographically after in vivo and in vitro binding experiments. Repeated swim stress decreased the in vivo binding in the hippocampus (p 125 I-iomazenil binding. These results suggest that we can investigate the pathophysiology of stress and anxiety with 123 I-iomazenil SPECT. Care must be taken concerning the effects of benzodiazepines. (author)

Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16

International Nuclear Information System (INIS)

Aguda, Adeleke Halilu; Sakwe, Amos Malle; Rask, Lars; Robinson, Robert Charles

2007-01-01

The crystallization and crystallographic data analysis of filamin repeats 14–16 are reported. Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14–16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 Å and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 Å, α = β = γ = 90°

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation.

Science.gov (United States)

Mueller, Sandra; Kleinau, Gunnar; Jaeschke, Holger; Paschke, Ralf; Krause, Gerd

2008-06-27

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae.

Science.gov (United States)

Zucchini, Laure; Mercy, Chryslène; Garcia, Pierre Simon; Cluzel, Caroline; Gueguen-Chaignon, Virginie; Galisson, Frédéric; Freton, Céline; Guiral, Sébastien; Brochier-Armanet, Céline; Gouet, Patrice; Grangeasse, Christophe

2018-02-01

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

International Nuclear Information System (INIS)

Ross, S.B.

1987-01-01

3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs

International Nuclear Information System (INIS)

Takagaki, Yoshio; Manley, J.L.; MacDonald, C.C.; Shenk, T.

1992-01-01

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. The authors have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-cross linked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. They have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long α-helix stabilized by salt bridges is predicted. An ∼270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (∼20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs

DNA dynamics is likely to be a factor in the genomic nucleotide repeats expansions related to diseases.

Directory of Open Access Journals (Sweden)

Boian S Alexandrov

Full Text Available Trinucleotide repeats sequences (TRS represent a common type of genomic DNA motif whose expansion is associated with a large number of human diseases. The driving molecular mechanisms of the TRS ongoing dynamic expansion across generations and within tissues and its influence on genomic DNA functions are not well understood. Here we report results for a novel and notable collective breathing behavior of genomic DNA of tandem TRS, leading to propensity for large local DNA transient openings at physiological temperature. Our Langevin molecular dynamics (LMD and Markov Chain Monte Carlo (MCMC simulations demonstrate that the patterns of openings of various TRSs depend specifically on their length. The collective propensity for DNA strand separation of repeated sequences serves as a precursor for outsized intermediate bubble states independently of the G/C-content. We report that repeats have the potential to interfere with the binding of transcription factors to their consensus sequence by altered DNA breathing dynamics in proximity of the binding sites. These observations might influence ongoing attempts to use LMD and MCMC simulations for TRS-related modeling of genomic DNA functionality in elucidating the common denominators of the dynamic TRS expansion mutation with potential therapeutic applications.

Paralogs hnRNP L and hnRNP LL exhibit overlapping but distinct RNA binding constraints.

Directory of Open Access Journals (Sweden)

Sarah A Smith

Full Text Available HnRNP (heterogeneous nuclear ribonucleoprotein proteins are a large family of RNA-binding proteins that regulate numerous aspects of RNA processing. Interestingly, several paralogous pairs of hnRNPs exist that exhibit similar RNA-binding specificity to one another, yet have non-redundant functional targets in vivo. In this study we systematically investigate the possibility that the paralogs hnRNP L and hnRNP LL have distinct RNA binding determinants that may underlie their lack of functional redundancy. Using a combination of RNAcompete and native gel analysis we find that while both hnRNP L and hnRNP LL preferentially bind sequences that contain repeated CA dinucleotides, these proteins differ in their requirement for the spacing of the CAs. Specifically, hnRNP LL has a more stringent requirement for a two nucleotide space between CA repeats than does hnRNP L, resulting in hnRNP L binding more promiscuously than does hnRNP LL. Importantly, this differential requirement for the spacing of CA dinucleotides explains the previously observed differences in the sensitivity of hnRNP L and LL to mutations within the CD45 gene. We suggest that overlapping but divergent RNA-binding preferences, as we show here for hnRNP L and hnRNP LL, may be commonplace among other hnRNP paralogs.

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

Science.gov (United States)

Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

2018-01-22

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

Energy Technology Data Exchange (ETDEWEB)

Ross, S.B.

1987-01-01

/sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

A protein engineered to bind uranyl selectively and with femtomolar affinity

Science.gov (United States)

Zhou, Lu; Bosscher, Mike; Zhang, Changsheng; Özçubukçu, Salih; Zhang, Liang; Zhang, Wen; Li, Charles J.; Liu, Jianzhao; Jensen, Mark P.; Lai, Luhua; He, Chuan

2014-03-01

Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM) however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

Tocotrienols are good adjuvants for developing cancer vaccines

International Nuclear Information System (INIS)

Abdul Hafid, Sitti Rahma; Radhakrishnan, Ammu Kutty; Nesaretnam, Kalanithi

2010-01-01

Dendritic cells (DCs) have the potential for cancer immunotherapy due to their ability to process and present antigens to T-cells and also in stimulating immune responses. However, DC-based vaccines have only exhibited minimal effectiveness against established tumours in mice and humans. The use of appropriate adjuvant enhances the efficacy of DC based cancer vaccines in treating tumours. In this study we have used tocotrienol-rich fraction (TRF), a non-toxic natural compound, as an adjuvant to enhance the effectiveness of DC vaccines in treating mouse mammary cancers. In the mouse model, six-week-old female BALB/c mice were injected subcutaneously with DC and supplemented with oral TRF daily (DC+TRF) and DC pulsed with tumour lysate from 4T1 cells (DC+TL). Experimental mice were also injected with DC pulsed with tumour lysate and supplemented daily with oral TRF (DC+TL+TRF) while two groups of animal which were supplemented daily with carrier oil (control) and with TRF (TRF). After three times vaccination, mice were inoculated with 4T1 cells in the mammary breast pad to induce tumour. Our study showed that TRF in combination with DC pulsed with tumour lysate (DC+TL+TRF) injected subcutaneously significantly inhibited the growth of 4T1 mammary tumour cells as compared to control group. Analysis of cytokines production from murine splenocytes showed significant increased productions of IFN-γ and IL-12 in experimental mice (DC+TL+TRF) compared to control, mice injected with DC without TRF, mice injected with DC pulsed with tumour lysate and mice supplemented with TRF alone. Higher numbers of cytotoxic T cells (CD8) and natural killer cells (NK) were observed in the peripheral blood of TRF adjuvanted DC pulsed tumour lysate mice. Our study show that TRF has the potential to be an adjuvant to augment DC based immunotherapy

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

Energy Technology Data Exchange (ETDEWEB)

Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

2007-01-01

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

Human pentraxin 3 binds to the complement regulator c4b-binding protein.

Directory of Open Access Journals (Sweden)

Anne Braunschweig

Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

Science.gov (United States)

Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

2015-01-01

Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

The wheat homolog of putative nucleotide-binding site-leucine-rich repeat resistance gene TaRGA contributes to resistance against powdery mildew.

Science.gov (United States)

Wang, Defu; Wang, Xiaobing; Mei, Yu; Dong, Hansong

2016-03-01

Powdery mildew, one of the most destructive wheat diseases worldwide, is caused by Blumeria graminis f. sp. tritici (Bgt), a fungal species with a consistently high mutation rate that makes individual resistance (R) genes ineffective. Therefore, effective resistance-related gene cloning is vital for breeding and studying the resistance mechanisms of the disease. In this study, a putative nucleotide-binding site-leucine-rich repeat (NBS-LRR) R gene (TaRGA) was cloned using a homology-based cloning strategy and analyzed for its effect on powdery mildew disease and wheat defense responses. Real-time reverse transcription-PCR (RT-PCR) analyses revealed that a Bgt isolate 15 and salicylic acid stimulation significantly induced TaRGA in the resistant variety. Furthermore, the silencing of TaRGA in powdery mildew-resistant plants increased susceptibility to Bgt15 and prompted conidia propagation at the infection site. However, the expression of TaRGA in leaf segments after single-cell transient expression assay highly increased the defense responses to Bgt15 by enhancing callose deposition and phenolic autofluorogen accumulation at the pathogen invading sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRGA-silenced plants and increased in the TaRGA-transient-overexpressing leaf segments. These results implied that the TaRGA gene positively regulates the defense response to powdery mildew disease in wheat.

Quantitative analysis and prediction of curvature in leucine-rich repeat proteins.

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Hindle, K Lauren; Bella, Jordi; Lovell, Simon C

2009-11-01

Leucine-rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein-protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature. It is this curvature that determines the shape and dimensions of the inner space available for ligand binding. Unfortunately, large-scale parameters such as the overall curvature of a protein domain are extremely difficult to predict. Here, we present a quantitative analysis of determinants of curvature of this family. Individual repeats typically range in length between 20 and 30 residues and have a variety of secondary structures on their convex side. The observed curvature of the LRR domains correlates poorly with the lengths of their individual repeats. We have, therefore, developed a scoring function based on the secondary structure of the convex side of the protein that allows prediction of the overall curvature with a high degree of accuracy. We also demonstrate the effectiveness of this method in selecting a suitable template for comparative modeling. We have developed an automated, quantitative protocol that can be used to predict accurately the curvature of leucine-rich repeat proteins of unknown structure from sequence alone. This protocol is available as an online resource at http://www.bioinf.manchester.ac.uk/curlrr/.

Unraveling the Role of RNA Mediated Toxicity of C9orf72 Repeats in C9-FTD/ALS

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Vijay Kumar

2017-12-01

Full Text Available The most frequent genetic cause of amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD is intronic hexanucleotide (G4C2 repeat expansions (HRE in the C9orf72 gene. The non-exclusive pathogenic mechanisms by which C9orf72 repeat expansions contribute to these neurological disorders include loss of C9orf72 function and gain-of-function determined by toxic RNA molecules and dipeptides repeats protein toxicity. The expanded repeats are transcribed bidirectionally and forms RNA foci in the central nervous system, and sequester key RNA-binding proteins (RBPs leading to impairment in RNA processing events. Many studies report widespread transcriptome changes in ALS carrying a C9orf72 repeat expansion. Here we review the contribution of RNA foci interaction with RBPs as well as transcriptome changes involved in the pathogenesis of C9orf72- associated FTD/ALS. These informations are essential to elucidate the pathology and therapeutic intervention of ALS and/or FTD.

The superfamily of C3b/C4b-binding proteins

DEFF Research Database (Denmark)

Kristensen, Torsten; D'Eustachio, P; Ogata, R T

1987-01-01

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing...... at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain...... of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively...

Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

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Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; Hong, Bum-Soo; Kim, Kyung-Phil; Shen, Yang; Lim, Kyung Jik; Mackenzie, Farrell; Tempel, Wolfram; Park, Hee-Won (SGC-Toronto); (PPCS); (Toronto)

2012-10-23

Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.

Tocotrienols are good adjuvants for developing cancer vaccines

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Radhakrishnan Ammu

2010-01-01

Full Text Available Abstract Background Dendritic cells (DCs have the potential for cancer immunotherapy due to their ability to process and present antigens to T-cells and also in stimulating immune responses. However, DC-based vaccines have only exhibited minimal effectiveness against established tumours in mice and humans. The use of appropriate adjuvant enhances the efficacy of DC based cancer vaccines in treating tumours. Methods In this study we have used tocotrienol-rich fraction (TRF, a non-toxic natural compound, as an adjuvant to enhance the effectiveness of DC vaccines in treating mouse mammary cancers. In the mouse model, six-week-old female BALB/c mice were injected subcutaneously with DC and supplemented with oral TRF daily (DC+TRF and DC pulsed with tumour lysate from 4T1 cells (DC+TL. Experimental mice were also injected with DC pulsed with tumour lysate and supplemented daily with oral TRF (DC+TL+TRF while two groups of animal which were supplemented daily with carrier oil (control and with TRF (TRF. After three times vaccination, mice were inoculated with 4T1 cells in the mammary breast pad to induce tumour. Results Our study showed that TRF in combination with DC pulsed with tumour lysate (DC+TL+TRF injected subcutaneously significantly inhibited the growth of 4T1 mammary tumour cells as compared to control group. Analysis of cytokines production from murine splenocytes showed significant increased productions of IFN-γ and IL-12 in experimental mice (DC+TL+TRF compared to control, mice injected with DC without TRF, mice injected with DC pulsed with tumour lysate and mice supplemented with TRF alone. Higher numbers of cytotoxic T cells (CD8 and natural killer cells (NK were observed in the peripheral blood of TRF adjuvanted DC pulsed tumour lysate mice. Conclusion Our study show that TRF has the potential to be an adjuvant to augment DC based immunotherapy.

Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

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He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

2000-08-01

Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

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Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

2017-10-01

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Wages and employment in a repeated game with revenue fluctuations

DEFF Research Database (Denmark)

Schultz, Christian

1997-01-01

Empirical investigations suggests that the real wage is surprisingly flat over the business cycle. This paper analyses a repeated game between a union and a firm which can contribute to explaining the flat wage. The parties cannot enter binding contracts, and revenue is fluctuating. The paper...... focuses on the best subgame-perfect equilibrium among those sharing the expected surplus in given fixed shares - e.g. equal shares. It is shown that (for moderate discount factors) this equilibrium has a more counter-cyclical wage, than what would be the case if the parties shared the surplus in each...

Transcriptome Profiling Reveals the Negative Regulation of Multiple Plant Hormone Signaling Pathways Elicited by Overexpression of C-Repeat Binding Factors

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Aixin Li

2017-09-01

Full Text Available C-repeat binding factors (CBF are a subfamily of AP2 transcription factors that play critical roles in the regulation of plant cold tolerance and growth in low temperature. In the present work, we sought to perform a detailed investigation into global transcriptional regulation of plant hormone signaling associated genes in transgenic plants engineered with CBF genes. RNA samples from Arabidopsis thaliana plants overexpressing two CBF genes, CBF2 and CBF3, were subjected to Illumina HiSeq 2000 RNA sequencing (RNA-Seq. Our results showed that more than half of the hormone associated genes that were differentially expressed in CBF2 or CBF3 transgenic plants were related to auxin signal transduction and metabolism. Most of these alterations in gene expression could lead to repression of auxin signaling. Accordingly, the IAA content was significantly decreased in young tissues of plants overexpressing CBF2 and CBF3 compared with wild type. In addition, genes associated with the biosynthesis of Jasmonate (JA and Salicylic acid (SA, as well as the signal sensing of Brassinolide (BR and SA, were down-regulated, while genes associated with Gibberellin (GA deactivation were up-regulated. In general, overexpression of CBF2 and CBF3 negatively affects multiple plant hormone signaling pathways in Arabidopsis. The transcriptome analysis using CBF2 and CBF3 transgenic plants provides novel and integrated insights into the interaction between CBFs and plant hormones, particularly the modulation of auxin signaling, which may contribute to the improvement of crop yields under abiotic stress via molecular engineering using CBF genes.

Regulation of the galanin system by repeated electroconvulsive seizures in mice

DEFF Research Database (Denmark)

Christiansen, S H; Woldbye, D P D

2010-01-01

of epileptic activity and depression, including the piriform cortex, hippocampal dentate gyrus, and amygdala. Likewise, GalR2 gene expression was up-regulated in both the central and the medial amygdala, whereas GalR1 gene expression showed a modest down-regulation in the medial amygdala. [(125) I......]galanin receptor binding in the piriform cortex, hippocampus, and amygdala was found to be significantly down-regulated. These data show that the galanin system is regulated by repeated ECS in a number of brain regions implicated in seizure regulation and depression. These changes may play a role...

Binding of the heterogeneous ribonucleoprotein K (hnRNP K to the Epstein-Barr virus nuclear antigen 2 (EBNA2 enhances viral LMP2A expression.

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Henrik Gross

Full Text Available The Epstein-Barr Virus (EBV -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively. EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2 Type 1. The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3 which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K. Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

Science.gov (United States)

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

2015-04-01

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue

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Aamand Jens

2011-01-01

Full Text Available Abstract The attachment of an antibody to an antigen-coated cantilever has been investigated by repeated experiments, using a cantilever-based detection system by Cantion A/S. The stress induced by the binding of a pesticide residue BAM (2,6 dichlorobenzamide immobilized on a cantilever surface to anti-BAM antibody is measured using the CantiLab4© system from Cantion A/S with four gold-coated cantilevers and piezo resistive readout. The detection mechanism is in principle label-free, but fluorescent-marked antibodies have been used to subsequently verify the binding on the cantilever surface. The bending and increase in mass of each cantilever has also been investigated using a light interferometer and a Doppler Vibrometer. The system has been analyzed during repeated measurements to investigate whether the CantiLab4© system is a suited platform for a pesticide assay system.

Crystal structure of CbpF, a bifunctional choline-binding protein and autolysis regulator from Streptococcus pneumoniae.

Science.gov (United States)

Molina, Rafael; González, Ana; Stelter, Meike; Pérez-Dorado, Inmaculada; Kahn, Richard; Morales, María; Moscoso, Miriam; Campuzano, Susana; Campillo, Nuria E; Mobashery, Shahriar; García, José L; García, Pedro; Hermoso, Juan A

2009-03-01

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline-binding proteins. The three-dimensional structure of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non-consensus choline-binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well-defined modules. The carboxy-terminal module is involved in cell wall binding and the amino-terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.

Steroid hormones affect binding of the sigma ligand 11C-SA4503 in tumour cells and tumour-bearing rats

International Nuclear Information System (INIS)

Rybczynska, Anna A.; Elsinga, Philip H.; Sijbesma, Jurgen W.; Jong, Johan R. de; Vries, Erik F. de; Dierckx, Rudi A.; Waarde, Aren van; Ishiwata, Kiichi

2009-01-01

Sigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist 11 C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels. 11 C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1 h or 5 min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone. Binding of 11 C-SA4503 to C6 cells was increased (∝50%) upon removal and decreased (∝60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC 50 progesterone 33 nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of 11 C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected. The binding of 11 C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids. (orig.)

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism

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Faraldo-Gómez, José D.

2017-01-01

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation. PMID:29167180

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism.

Science.gov (United States)

Ficici, Emel; Faraldo-Gómez, José D; Jennings, Michael L; Forrest, Lucy R

2017-12-04

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation. © 2017 Ficici et al.

[open quotes]Cryptic[close quotes] repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: Analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes

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Gostout, B.; Qiang Liu; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

1993-06-01

Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are [open quotes]cryptic[close quotes] repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29--40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differently distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. The data suggest how simple tandem repeats could evolve from cryptic repeats. 18 refs., 3 figs., 6 tabs.

LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

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Jean-Marc Taymans

Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

Repeating and non-repeating fast radio bursts from binary neutron star mergers

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Yamasaki, Shotaro; Totani, Tomonori; Kiuchi, Kenta

2018-04-01

Most fast radio bursts (FRB) do not show evidence of repetition, and such non-repeating FRBs may be produced at the time of a merger of binary neutron stars (BNS), provided that the BNS merger rate is close to the high end of the currently possible range. However, the merger environment is polluted by dynamical ejecta, which may prohibit the radio signal from propagating. We examine this by using a general-relativistic simulation of a BNS merger, and show that the ejecta appears about 1 ms after the rotation speed of the merged star becomes the maximum. Therefore there is a time window in which an FRB signal can reach outside, and the short duration of non-repeating FRBs can be explained by screening after ejecta formation. A fraction of BNS mergers may leave a rapidly rotating and stable neutron star, and such objects may be the origin of repeating FRBs like FRB 121102. We show that a merger remnant would appear as a repeating FRB on a time scale of ˜1-10 yr, and expected properties are consistent with the observations of FRB 121102. We construct an FRB rate evolution model that includes these two populations of repeating and non-repeating FRBs from BNS mergers, and show that the detection rate of repeating FRBs relative to non-repeating ones rapidly increases with improving search sensitivity. This may explain why only the repeating FRB 121102 was discovered by the most sensitive FRB search with Arecibo. Several predictions are made, including the appearance of a repeating FRB 1-10 yr after a BNS merger that is localized by gravitational waves and subsequent electromagnetic radiation.

The effect of a DeltaK280 mutation on the unfolded state of a microtubule-binding repeat in Tau.

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Austin Huang

Full Text Available Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer's disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW, samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2 in wild-type (WT tau and a DeltaK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and DeltaK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of beta-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.

The telomere length dynamic and methods of its assessment.

Science.gov (United States)

Lin, Kah-Wai; Yan, Ju

2005-01-01

Human telomeres are composed of long repeating sequences of TTAGGG, associated with a variety of telomere-binding proteins. Its function as an end-protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation. Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevent the loss of telomeres due to the end replication problems. However, telomerase activity is detected at low level in somatic cells and high level in embryonic stem cells and tumor cells. It confers immortality to embryonic stem cells and tumor cells. In most tumor cells, telomeres are extremely short and stable. Telomere length is an important indicator of the telomerase activity in tumor cells and it may be used in the prognosis of malignancy. Thus, the assessment of telomeres length is of great experimental and clinical significance. This review describes the role of telomere and telomerase in cancer pathogenesis and the dynamics of the telomeres length in different cell types. The various methods of measurement of telomeres length, i.e. southern blot, hybridization protection assay, fluorescence in situ hybridization, primed in situ, quantitative PCR and single telomere length analysis are discussed. The principle and comparative evaluation of these methods are reviewed. The detection of G-strand overhang by telomeric-oligonucleotide ligation assay, primer extension/nick translation assay and electron microscopy are briefly discussed.

Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip.

Science.gov (United States)

Nelson, Gregory M; Huffman, Holly; Smith, David F

2003-01-01

Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function.

The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

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Henry A Choy

Full Text Available Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis

A Smart DNA Tweezer for Detection of Human Telomerase Activity.

Science.gov (United States)

Xu, Xiaowen; Wang, Lei; Li, Kan; Huang, Qihong; Jiang, Wei

2018-03-06

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) n . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci

DEFF Research Database (Denmark)

Alkhnbashi, Omer S.; Costa, Fabrizio; Shah, Shiraz Ali

2014-01-01

Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR...... array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci...... by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection...

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

Science.gov (United States)

Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-04-01

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

Science.gov (United States)

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

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Toshitsugu Fujita

2015-09-01

Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

The Plant Leucine-Rich Repeat Receptor-Like Kinase PSY1R from Head to Toe

DEFF Research Database (Denmark)

Oehlenschlæger, Christian Berg

PSY1R belongs to the family of plant leucine-rich repeat receptor-like kinases that play important roles in processes such as growth regulation and plant immunity response. PSY1R was proposed to be the receptor of the plant peptide hormone PSY1 which promotes cell expansion. PSY1R was furthermore...... is activated. This work provides the first study of the direct interaction between PSY1R and the peptide ligand PSY1. The binding was evaluated both for full length PSY1R expressed in plants and for the isolated extracellular domain expressed in insect cells. PSY1 binds to the extracellular domain of PSY1R...... shown to phosphorylate and regulate the activity of the plasma membrane localized H+-ATPase, AHA2. While the mechanism of PSY1R-mediated AHA2 phosphorylation has previously been studied in detail, little is known about how PSY1R binds PSY1 peptide ligand and how the intracellular PSY1R kinase domain...

The Influence of the Terrestrial Reference Frame on Studies of Sea Level Change

Science.gov (United States)

Nerem, R. S.; Bar-Sever, Y. E.; Haines, B. J.; Desai, S.; Heflin, M. B.

2015-12-01

The terrestrial reference frame (TRF) provides the foundation for the accurate monitoring of sea level using both ground-based (tide gauges) and space-based (satellite altimetry) techniques. For the latter, tide gauges are also used to monitor drifts in the satellite instruments over time. The accuracy of the terrestrial reference frame (TRF) is thus a critical component for both types of sea level measurements. The TRF is central to the formation of geocentric sea-surface height (SSH) measurements from satellite altimeter data. The computed satellite orbits are linked to a particular TRF via the assumed locations of the ground-based tracking systems. The manner in which TRF errors are expressed in the orbit solution (and thus SSH) is not straightforward, and depends on the models of the forces underlying the satellite's motion. We discuss this relationship, and provide examples of the systematic TRF-induced errors in the altimeter derived sea-level record. The TRF is also crucial to the interpretation of tide-gauge measurements, as it enables the separation of vertical land motion from volumetric changes in the water level. TRF errors affect tide gauge measurements through GNSS estimates of the vertical land motion at each tide gauge. This talk will discuss the current accuracy of the TRF and how errors in the TRF impact both satellite altimeter and tide gauge sea level measurements. We will also discuss simulations of how the proposed Geodetic Reference Antenna in SPace (GRASP) satellite mission could reduce these errors and revolutionize how reference frames are computed in general.

Partial genetic deletion of neuregulin 1 and adolescent stress interact to alter NMDA receptor binding in the medial prefrontal cortex

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Tariq Waseem Chohan

2014-09-01

Full Text Available Schizophrenia is thought to arise due to a complex interaction between genetic and environmental factors during early neurodevelopment. We have recently shown that partial genetic deletion of the schizophrenia susceptibility gene neuregulin 1 (Nrg1 and adolescent stress interact to disturb sensorimotor gating, neuroendocrine activity and dendritic morphology in mice. Both stress and Nrg1 may have converging effects upon N-methyl-D-aspartate receptors (NMDARs which are implicated in the pathogenesis of schizophrenia, sensorimotor gating and dendritic spine plasticity. Using an identical repeated restraint stress paradigm to our previous study, here we determined NMDAR binding across various brain regions in adolescent Nrg1 heterozygous (HET and wild-type (WT mice using [3H] MK-801 autoradiography. Repeated restraint stress increased NMDAR binding in the ventral part of the lateral septum (LSV and the dentate gyrus (DG of the hippocampus irrespective of genotype. Partial genetic deletion of Nrg1 interacted with adolescent stress to promote an altered pattern of NMDAR binding in the infralimbic (IL subregion of the medial prefrontal cortex. In the IL, whilst stress tended to increase NMDAR binding in WT mice, it decreased binding in Nrg1 HET mice. However in the DG, stress selectively increased the expression of NMDAR binding in Nrg1 HET mice but not WT mice. These results demonstrate a Nrg1-stress interaction during adolescence on NMDAR binding in the medial prefrontal cortex.

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin*♦

Science.gov (United States)

Bensing, Barbara A.; Loukachevitch, Lioudmila V.; McCulloch, Kathryn M.; Yu, Hai; Vann, Kendra R.; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M.; Iverson, T. M.

2016-01-01

Streptococcus sanguinis is a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets. S. sanguinis expresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site. PMID:26833566

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression

Energy Technology Data Exchange (ETDEWEB)

Siddiqui, Shabeena [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India); Ahsan, Haseeb [Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi 110025 (India); Khan, Mohammad Rashid [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India); Siddiqui, Waseem A., E-mail: wasiddiqui01@gmail.com [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India)

2013-12-01

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175–200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. - Highlights: • The nephroprotective effect of TRF in type-2 diabetic rats was investigated. • Treatment with TRF improved glycemic status, lipid profile and renal functions in rats

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression

International Nuclear Information System (INIS)

Siddiqui, Shabeena; Ahsan, Haseeb; Khan, Mohammad Rashid; Siddiqui, Waseem A.

2013-01-01

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175–200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. - Highlights: • The nephroprotective effect of TRF in type-2 diabetic rats was investigated. • Treatment with TRF improved glycemic status, lipid profile and renal functions in rats

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-β expression

Energy Technology Data Exchange (ETDEWEB)

Siddiqui, Shabeena [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India); Ahsan, Haseeb [Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi 110025 (India); Khan, Mohammad Rashid [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India); Siddiqui, Waseem A., E-mail: wasiddiqui01@gmail.com [Department of Biochemistry, Lipid Metabolism Laboratory, Jamia Hamdard (Hamdard University), New Delhi 110062 (India)

2013-12-01

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175–200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-β, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-β, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-β (TGF-β) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-β to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF. - Highlights: • The nephroprotective effect of TRF in type-2 diabetic rats was investigated. • Treatment with TRF improved glycemic status, lipid profile and renal functions in rats

Preferential reduction of binding of 125I-iodopindolol to beta-1 adrenoceptors in the amygdala of rat after antidepressant treatments

International Nuclear Information System (INIS)

Ordway, G.A.; Gambarana, C.; Tejani-Butt, S.M.; Areso, P.; Hauptmann, M.; Frazer, A.

1991-01-01

This study utilized quantitative receptor autoradiography to examine the effects of repeated administration of antidepressants to rats on the binding of the beta adrenoceptor antagonist, 125 I-iodopindolol ( 125 I-IPIN) to either beta-1 or beta-2 adrenoceptors in various regions of brain. Antidepressants were selected to represent various chemical and pharmacological classes including tricyclic compounds (desipramine and protriptyline), monoamine oxidase inhibitors (clorgyline, phenelzine and tranylcypromine), atypical antidepressants (mianserin and trazodone) and selective inhibitors of the uptake of serotonin (citalopram and sertraline). Additionally, rats were treated with various psychotropic drugs that lack antidepressant efficacy (cocaine, deprenyl, diazepam and haloperidol). Repeated treatment of rats with desipramine, protriptyline, clorgyline, phenelzine, tranylcypromine or mianserin reduced the binding of 125 I-IPIN to beta-1 adrenoceptors in many brain areas. Only in the basolateral and lateral nuclei of the amygdala did all six of these antidepressants significantly reduce 125 I-IPIN binding to beta-1 adrenoceptors. In these amygdaloid nuclei, the magnitude of the reduction in the binding of 125 I-IPIN caused by each of these drugs was comparable to or greater than the reduction in binding produced in any other region of brain. Reductions of binding of 125 I-IPIN after antidepressant treatments were not consistently observed in the cortex, the area of brain examined most often in homogenate binding studies. Only the monoamine oxidase inhibitors caused reductions in the binding of 125 I-IPIN to beta-2 adrenoceptors, and this effect was generally localized to the amygdala and hypothalamus

A Unified Model for Repeating and Non-repeating Fast Radio Bursts

International Nuclear Information System (INIS)

Bagchi, Manjari

2017-01-01

The model that fast radio bursts (FRBs) are caused by plunges of asteroids onto neutron stars can explain both repeating and non-repeating bursts. If a neutron star passes through an asteroid belt around another star, there would be a series of bursts caused by a series of asteroid impacts. Moreover, the neutron star would cross the same belt repetitively if it were in a binary with the star hosting the asteroid belt, leading to a repeated series of bursts. I explore the properties of neutron star binaries that could lead to the only known repeating FRB so far (FRB121102). In this model, the next two epochs of bursts are expected around 2017 February 27 and 2017 December 18. On the other hand, if the asteroid belt is located around the neutron star itself, then a chance fall of an asteroid from that belt onto the neutron star would lead to a non-repeating burst. Even a neutron star grazing an asteroid belt can lead to a non-repeating burst caused by just one asteroid plunge during the grazing. This is possible even when the neutron star is in a binary with the asteroid-hosting star, if the belt and the neutron star orbit are non-coplanar.

A Unified Model for Repeating and Non-repeating Fast Radio Bursts

Energy Technology Data Exchange (ETDEWEB)

Bagchi, Manjari, E-mail: manjari@imsc.res.in [The Institute of Mathematical Sciences (IMSc-HBNI), 4th Cross Road, CIT Campus, Taramani, Chennai 600113 (India)

2017-04-01

The model that fast radio bursts (FRBs) are caused by plunges of asteroids onto neutron stars can explain both repeating and non-repeating bursts. If a neutron star passes through an asteroid belt around another star, there would be a series of bursts caused by a series of asteroid impacts. Moreover, the neutron star would cross the same belt repetitively if it were in a binary with the star hosting the asteroid belt, leading to a repeated series of bursts. I explore the properties of neutron star binaries that could lead to the only known repeating FRB so far (FRB121102). In this model, the next two epochs of bursts are expected around 2017 February 27 and 2017 December 18. On the other hand, if the asteroid belt is located around the neutron star itself, then a chance fall of an asteroid from that belt onto the neutron star would lead to a non-repeating burst. Even a neutron star grazing an asteroid belt can lead to a non-repeating burst caused by just one asteroid plunge during the grazing. This is possible even when the neutron star is in a binary with the asteroid-hosting star, if the belt and the neutron star orbit are non-coplanar.

Repeatability of Cryogenic Multilayer Insulation

Science.gov (United States)

Johnson, W. L.; Vanderlaan, M.; Wood, J. J.; Rhys, N. O.; Guo, W.; Van Sciver, S.; Chato, D. J.

2017-12-01

Due to the variety of requirements across aerospace platforms, and one off projects, the repeatability of cryogenic multilayer insulation (MLI) has never been fully established. The objective of this test program is to provide a more basic understanding of the thermal performance repeatability of MLI systems that are applicable to large scale tanks. There are several different types of repeatability that can be accounted for: these include repeatability between identical blankets, repeatability of installation of the same blanket, and repeatability of a test apparatus. The focus of the work in this report is on the first two types of repeatability. Statistically, repeatability can mean many different things. In simplest form, it refers to the range of performance that a population exhibits and the average of the population. However, as more and more identical components are made (i.e. the population of concern grows), the simple range morphs into a standard deviation from an average performance. Initial repeatability testing on MLI blankets has been completed at Florida State University. Repeatability of five Glenn Research Center (GRC) provided coupons with 25 layers was shown to be +/- 8.4% whereas repeatability of repeatedly installing a single coupon was shown to be +/- 8.0%. A second group of 10 coupons has been fabricated by Yetispace and tested by Florida State University, the repeatability between coupons has been shown to be +/- 15-25%. Based on detailed statistical analysis, the data has been shown to be statistically significant.

Src binds cortactin through an SH2 domain cystine-mediated linkage

Science.gov (United States)

Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

2012-01-01

Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

Src binds cortactin through an SH2 domain cystine-mediated linkage.

Science.gov (United States)

Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

2012-12-15

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

Deployment Repeatability

Science.gov (United States)

2016-04-01

evaluating the deployment repeatability builds upon the testing or analysis of deployment kinematics (Chapter 6) and adds repetition. Introduction...material yield or failure during a test. For the purposes of this chapter, zero shift will refer to permanent changes in the structure, while reversible ...the content of other chapters in this book: Gravity Compensation (Chapter 4) and Deployment Kinematics and Dynamics (Chapter 6). Repeating the

Differences in Binding and Monitoring Mechanisms Contribute to Lifespan Age Differences in False Memory

Science.gov (United States)

Fandakova, Yana; Shing, Yee Lee; Lindenberger, Ulman

2013-01-01

Based on a 2-component framework of episodic memory development across the lifespan (Shing & Lindenberger, 2011), we examined the contribution of memory-related binding and monitoring processes to false memory susceptibility in childhood and old age. We administered a repeated continuous recognition task to children (N = 20, 10-12 years),…

The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

DEFF Research Database (Denmark)

Behrendt, N; Ploug, M; Patthy, L

1991-01-01

with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

APE1 incision activity at abasic sites in tandem repeat sequences.

Science.gov (United States)

Li, Mengxia; Völker, Jens; Breslauer, Kenneth J; Wilson, David M

2014-05-29

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains. Published by Elsevier Ltd.

P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

OpenAIRE

Pal, S K; Chattoraj, D K

1988-01-01

The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

Telomere binding protein TRB1 is associated with promoters of translation machinery genes in vivo

Czech Academy of Sciences Publication Activity Database

Schrumpfová, P.; Vychodilová, I.; Hapala, J.; Schorová, Š.; Dvořáček, Vojtěch; Fajkus, Jiří

2016-01-01

Roč. 90, 1-2 (2016), s. 189-206 ISSN 0167-4412 R&D Projects: GA ČR(CZ) GA13-06943S Institutional support: RVO:68081707 Keywords : Telomere repeat binding * ChIP-seq * Arabidopsis thaliana Subject RIV: BO - Biophysics Impact factor: 3.356, year: 2016

Development of a Conceptual Etiological Model of Treatment Regimen Fatigue Among Patients Engaged in HIV Care: A Qualitative Study.

Science.gov (United States)

Claborn, Kasey; Miller, Mary Beth; Meier, Ellen; Carbone, Sofia

Treatment regimen fatigue (TRF) is a decreased desire and motivation to maintain vigilance in adhering to treatment, and little is known about TRF in people living with HIV. We aimed to develop a conceptual framework of TRF. Five focus groups were conducted in 2014. Eligible participants were (a) HIV infected, (b) at least 18 years of age, (c) prescribed antiretroviral therapy, and (d) fluent in English. Data were analyzed using thematic analysis. Analyses revealed these themes: patient experiences of TRF; etiological factors at the systems, provider, and patient levels; strategies to manage TRF; consequences of TRF; and protective factors that prevent the occurrence of TRF. The results provided a conceptual framework for future investigations to build on in an effort to improve adherence and retention in HIV care. Study results indicate avenues for intervention at multiple levels (systems, provider, and patient) to reduce treatment burden and improve patient resources and capacity. Copyright © 2017 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.

Reconfigurable multiport EPON repeater

Science.gov (United States)

Oishi, Masayuki; Inohara, Ryo; Agata, Akira; Horiuchi, Yukio

2009-11-01

An extended reach EPON repeater is one of the solutions to effectively expand FTTH service areas. In this paper, we propose a reconfigurable multi-port EPON repeater for effective accommodation of multiple ODNs with a single OLT line card. The proposed repeater, which has multi-ports in both OLT and ODN sides, consists of TRs, BTRs with the CDR function and a reconfigurable electrical matrix switch, can accommodate multiple ODNs to a single OLT line card by controlling the connection of the matrix switch. Although conventional EPON repeaters require full OLT line cards to accommodate subscribers from the initial installation stage, the proposed repeater can dramatically reduce the number of required line cards especially when the number of subscribers is less than a half of the maximum registerable users per OLT. Numerical calculation results show that the extended reach EPON system with the proposed EPON repeater can save 17.5% of the initial installation cost compared with a conventional repeater, and can be less expensive than conventional systems up to the maximum subscribers especially when the percentage of ODNs in lightly-populated areas is higher.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants

DEFF Research Database (Denmark)

Rougemaille, Mathieu; Gudipati, Rajani K; Olesen, Jens Raabjerg

2007-01-01

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access...... is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein...

Steroid hormones affect binding of the sigma ligand {sup 11}C-SA4503 in tumour cells and tumour-bearing rats

Energy Technology Data Exchange (ETDEWEB)

Rybczynska, Anna A.; Elsinga, Philip H.; Sijbesma, Jurgen W.; Jong, Johan R. de; Vries, Erik F. de; Dierckx, Rudi A.; Waarde, Aren van [University of Groningen, Nuclear Medicine and Molecular Imaging, University of Groningen Medical Center, Groningen (Netherlands); Ishiwata, Kiichi [Tokyo Metropolitan Institute of Gerontology, Positron Medical Center, Tokyo (Japan)

2009-07-15

Sigma receptors are implicated in memory and cognitive functions, drug addiction, depression and schizophrenia. In addition, sigma receptors are strongly overexpressed in many tumours. Although the natural ligands are still unknown, steroid hormones are potential candidates. Here, we examined changes in binding of the sigma-1 agonist {sup 11}C-SA4503 in C6 glioma cells and in living rats after modification of endogenous steroid levels. {sup 11}C-SA4503 binding was assessed in C6 monolayers by gamma counting and in anaesthetized rats by microPET scanning. C6 cells were either repeatedly washed and incubated in steroid-free medium or exposed to five kinds of exogenous steroids (1 h or 5 min before tracer addition, respectively). Tumour-bearing male rats were repeatedly treated with pentobarbital (a condition known to result in reduction of endogenous steroid levels) or injected with progesterone. Binding of {sup 11}C-SA4503 to C6 cells was increased ({proportional_to}50%) upon removal and decreased ({proportional_to}60%) upon addition of steroid hormones (rank order of potency: progesterone > allopregnanolone = testosterone = androstanolone > dehydroepiandrosterone-3-sulphate, IC{sub 50} progesterone 33 nM). Intraperitoneally administered progesterone reduced tumour uptake and tumour-to-muscle contrast (36%). Repeated treatment of animals with pentobarbital increased the PET standardized uptake value of {sup 11}C-SA4503 in tumour (16%) and brain (27%), whereas the kinetics of blood pool radioactivity was unaffected. The binding of {sup 11}C-SA4503 is sensitive to steroid competition. Since not only increases but also decreases of steroid levels affect ligand binding, a considerable fraction of the sigma-1 receptor population in cultured tumour cells or tumour-bearing animals is normally occupied by endogenous steroids. (orig.)

Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes.

Science.gov (United States)

Hashiguchi, Y; Lee, J M; Shiraishi, M; Komatsu, S; Miki, S; Shimasaki, Y; Mochioka, N; Kusakabe, T; Oshima, Y

2015-05-01

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

Comparative Pharmacokinetics of Cefquinome (Cobactan 2.5% following Repeated Intramuscular Administrations in Sheep and Goats

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Mohamed El-Hewaity

2014-01-01

Full Text Available The comparative pharmacokinetic profile of cefquinome was studied in sheep and goats following repeated intramuscular (IM administrations of 2 mg/kg body weight. Cefquinome concentrations in serum were determined by microbiological assay technique using Micrococcus luteus (ATCC 9341 as test organism. Following intramuscular injection of cefquinome in sheep and goats, the disposition curves were best described by two-compartment open model in both sheep and goats. The pharmacokinetics of cefquinome did not differ significantly between sheep and goats; similar intramuscular dose rate of cefquinome should therefore be applicable to both species. On comparing the data of serum levels of repeated intramuscular injections with first intramuscular injection, it was revealed that repeated intramuscular injections of cefquinome have cumulative effect in both species sheep and goats. The in vitro serum protein-binding tendency was 15.65% in sheep and 14.42% in goats. The serum concentrations of cefquinome along 24 h after injection in this study were exceeding the MICs of different susceptible microorganisms responsible for serious disease problems. These findings indicate successful use of cefquinome in sheep and goats.

[11C]Harmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification.

Science.gov (United States)

Zanderigo, Francesca; D'Agostino, Alexandra E; Joshi, Nandita; Schain, Martin; Kumar, Dileep; Parsey, Ramin V; DeLorenzo, Christine; Mann, J John

2018-02-08

Inhibition of the isoform A of monoamine oxidase (MAO-A), a mitochondrial enzyme catalyzing deamination of monoamine neurotransmitters, is useful in treatment of depression and anxiety disorders. [ 11 C]harmine, a MAO-A PET radioligand, has been used to study mood disorders and antidepressant treatment. However, [ 11 C]harmine binding test-retest characteristics have to date only been partially investigated. Furthermore, since MAO-A is ubiquitously expressed, no reference region is available, thus requiring arterial blood sampling during PET scanning. Here, we investigate [ 11 C]harmine binding measurements test-retest properties; assess effects of using a minimally invasive input function estimation on binding quantification and repeatability; and explore binding potentials estimation using a reference region-free approach. Quantification of [ 11 C]harmine distribution volume (V T ) via kinetic models and graphical analyses was compared based on absolute test-retest percent difference (TRPD), intraclass correlation coefficient (ICC), and identifiability. The optimal procedure was also used with a simultaneously estimated input function in place of the measured curve. Lastly, an approach for binding potentials quantification in absence of a reference region was evaluated. [ 11 C]harmine V T estimates quantified using arterial blood and kinetic modeling showed average absolute TRPD values of 7.7 to 15.6 %, and ICC values between 0.56 and 0.86, across brain regions. Using simultaneous estimation (SIME) of input function resulted in V T estimates close to those obtained using arterial input function (r = 0.951, slope = 1.073, intercept = - 1.037), with numerically but not statistically higher test-retest difference (range 16.6 to 22.0 %), but with overall poor ICC values, between 0.30 and 0.57. Prospective studies using [ 11 C]harmine are possible given its test-retest repeatability when binding is quantified using arterial blood. Results with SIME of

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding

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Corbo Laura

2001-11-01

Full Text Available Abstract Background The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. Results Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals, which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. Conclusions We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

Science.gov (United States)

Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

2011-04-01

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Origin and global diversification patterns of tropical rain forests: inferences from a complete genus-level phylogeny of palms

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Couvreur Thomas LP

2011-06-01

Full Text Available Abstract Background Understanding how biodiversity is shaped through time is a fundamental question in biology. Even though tropical rain forests (TRF represent the most diverse terrestrial biomes on the planet, the timing, location and mechanisms of their diversification remain poorly understood. Molecular phylogenies are valuable tools for exploring these issues, but to date most studies have focused only on recent time scales, which minimises their explanatory potential. In order to provide a long-term view of TRF diversification, we constructed the first complete genus-level dated phylogeny of a largely TRF-restricted plant family with a known history dating back to the Cretaceous. Palms (Arecaceae/Palmae are one of the most characteristic and ecologically important components of TRF worldwide, and represent a model group for the investigation of TRF evolution. Results We provide evidence that diversification of extant lineages of palms started during the mid-Cretaceous period about 100 million years ago. Ancestral biome and area reconstructions for the whole family strongly support the hypothesis that palms diversified in a TRF-like environment at northern latitudes. Finally, our results suggest that palms conform to a constant diversification model (the 'museum' model or Yule process, at least until the Neogene, with no evidence for any change in diversification rates even through the Cretaceous/Paleogene mass extinction event. Conclusions Because palms are restricted to TRF and assuming biome conservatism over time, our results suggest the presence of a TRF-like biome in the mid-Cretaceous period of Laurasia, consistent with controversial fossil evidence of the earliest TRF. Throughout its history, the TRF biome is thought to have been highly dynamic and to have fluctuated greatly in extent, but it has persisted even during climatically unfavourable periods. This may have allowed old lineages to survive and contribute to the steady

Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum

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Sam Manna

2013-01-01

Full Text Available Pentatricopeptide repeat (PPR proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protist Dictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

Cassava C-repeat binding factor 1 gene responds to low temperature and enhances cold tolerance when overexpressed in Arabidopsis and cassava.

Science.gov (United States)

An, Dong; Ma, Qiuxiang; Wang, Hongxia; Yang, Jun; Zhou, Wenzhi; Zhang, Peng

2017-05-01

Cassava MeCBF1 is a typical CBF transcription factor mediating cold responses but its low expression in apical buds along with a retarded response cause inefficient upregulation of downstream cold-related genes, rendering cassava chilling-sensitive. Low temperature is a major abiotic stress factor affecting survival, productivity and geographic distribution of important crops worldwide. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important regulators of abiotic stress response in plants. In this study, MeCBF1, a CBF-like gene, was identified in the tropical root crop cassava (Manihot esculenta Crantz). The MeCBF1 encodes a protein that shares strong homology with DREB1As/CBFs from Arabidopsis as well as other species. The MeCBF1 was localized to the nucleus and is mainly expressed in stem and mature leaves, but not in apical buds or stem cambium. MeCBF1 expression was not only highly responsive to cold, but also significantly induced by salt, PEG and ABA treatment. Several stress-associated cis-elements were found in its promoter region, e.g., ABRE-related, MYC recognition sites, and MYB responsive element. Compared with AtCBF1, the MeCBF1 expression induced by cold in cassava was retarded and upregulated only after 4 h, which was also confirmed by its promoter activity. Overexpression of MeCBF1 in transgenic Arabidopsis and cassava plants conferred enhanced crytolerance. The CBF regulon was smaller and not entirely co-regulated with MeCBF1 expression in overexpressed cassava. The retarded MeCBF1 expression in response to cold and attenuated CBF-regulon might lead cassava to chilling sensitivity.

Common carotid artery intima-media thickness is as good as carotid intima-media thickness of all carotid artery segments in improving prediction of coronary heart disease risk in the Atherosclerosis Risk in Communities (ARIC) study.

Science.gov (United States)

Nambi, Vijay; Chambless, Lloyd; He, Max; Folsom, Aaron R; Mosley, Tom; Boerwinkle, Eric; Ballantyne, Christie M

2012-01-01

Carotid intima-media thickness (CIMT) and plaque information can improve coronary heart disease (CHD) risk prediction when added to traditional risk factors (TRF). However, obtaining adequate images of all carotid artery segments (A-CIMT) may be difficult. Of A-CIMT, the common carotid artery intima-media thickness (CCA-IMT) is relatively more reliable and easier to measure. We evaluated whether CCA-IMT is comparable to A-CIMT when added to TRF and plaque information in improving CHD risk prediction in the Atherosclerosis Risk in Communities (ARIC) study. Ten-year CHD risk prediction models using TRF alone, TRF + A-CIMT + plaque, and TRF + CCA-IMT + plaque were developed for the overall cohort, men, and women. The area under the receiver operator characteristic curve (AUC), per cent individuals reclassified, net reclassification index (NRI), and model calibration by the Grønnesby-Borgan test were estimated. There were 1722 incident CHD events in 12 576 individuals over a mean follow-up of 15.2 years. The AUC for TRF only, TRF + A-CIMT + plaque, and TRF + CCA-IMT + plaque models were 0.741, 0.754, and 0.753, respectively. Although there was some discordance when the CCA-IMT + plaque- and A-CIMT + plaque-based risk estimation was compared, the NRI and clinical NRI (NRI in the intermediate-risk group) when comparing the CIMT models with TRF-only model, per cent reclassified, and test for model calibration were not significantly different. Coronary heart disease risk prediction can be improved by adding A-CIMT + plaque or CCA-IMT + plaque information to TRF. Therefore, evaluating the carotid artery for plaque presence and measuring CCA-IMT, which is easier and more reliable than measuring A-CIMT, provide a good alternative to measuring A-CIMT for CHD risk prediction.

Plant phylogeny as a window on the evolution of hyperdiversity in the tropical rainforest biome.

Science.gov (United States)

Eiserhardt, Wolf L; Couvreur, Thomas L P; Baker, William J

2017-06-01

I. II. III. IV. V. VI. VII. VIII. IX. References SUMMARY: Tropical rainforest (TRF) is the most species-rich terrestrial biome on Earth, harbouring just under half of the world's plant species in c. 7% of the land surface. Phylogenetic trees provide important insights into mechanisms underpinning TRF hyperdiversity that are complementary to those obtained from the fossil record. Phylogenetic studies of TRF plant diversity have mainly focused on whether this biome is an evolutionary 'cradle' or 'museum', emphasizing speciation and extinction rates. However, other explanations, such as biome age, immigration and ecological limits, must also be considered. We present a conceptual framework for addressing the drivers of TRF diversity, and review plant studies that have tested them with phylogenetic data. Although surprisingly few in number, these studies point to old age of TRF, low extinction and high speciation rates as credible drivers of TRF hyperdiversity. There is less evidence for immigration and ecological limits, but these cannot be dismissed owing to the limited number of studies. Rapid methodological developments in DNA sequencing, macroevolutionary analysis and the integration of phylogenetics with other disciplines may improve our grasp of TRF hyperdiversity in the future. However, such advances are critically dependent on fundamental systematic research, yielding numerous, additional, well-sampled phylogenies of TRF lineages. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

The leucine-rich repeat structure.

Science.gov (United States)

Bella, J; Hindle, K L; McEwan, P A; Lovell, S C

2008-08-01

The leucine-rich repeat is a widespread structural motif of 20-30 amino acids with a characteristic repetitive sequence pattern rich in leucines. Leucine-rich repeat domains are built from tandems of two or more repeats and form curved solenoid structures that are particularly suitable for protein-protein interactions. Thousands of protein sequences containing leucine-rich repeats have been identified by automatic annotation methods. Three-dimensional structures of leucine-rich repeat domains determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. As the essential structural principles become well established, the leucine-rich repeat architecture is emerging as an attractive framework for structural prediction and protein engineering. This review presents an update of the current understanding of leucine-rich repeat structure at the primary, secondary, tertiary and quaternary levels and discusses specific examples from recently determined three-dimensional structures.

Crystal structures of the human G3BP1 NTF2-like domain visualize FxFG Nup Repeat Specificity

DEFF Research Database (Denmark)

Vognsen, Tina Reinholdt; Möller, Ingvar Rúnar; Kristensen, Ole

2013-01-01

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat...... peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded...

Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

Science.gov (United States)

Wu, Enqi; Peng, Yuan; Zhang, Xihao; Bai, Jialei; Song, Yanqiu; He, Houluo; Fan, Longxing; Qu, Xiaochen; Gao, Zhixian; Liu, Ying; Ning, Baoan

2017-02-22

A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10 -3 ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.

Radioactive labelling of peptidic hormones

International Nuclear Information System (INIS)

Fromageot, P.; Pradelles, P.; Morgat, J.L.; Levine, H.

1976-01-01

The labelling of peptidic hormones requires stability, specificity and sensitivity of the label. Introduction of a radioactive atome is one way to satisfy these criteria. Several processes have been described to prepare radioactive TRF: synthesis of the peptide with labelled aminoacids or introduction of the label into the hormone. In that approach, tritium can be substituted in the imidazole ring, via precursors activating the proper carbon. Monoiodo TRF leads essentially to tritium labelling of the 5 positions whereas monoazo TRF allows the preparation of 3 H TRF labelled in the 2 positions. Di-substituted TRF leads to labelling into the 2 and 5 carbons. Labelled analogs of TRF can be prepared with labelled iodine; further developments of peptide labelling, will be presented. In particular, the homolytic scission of the C-iodine, bond by photochemical activation. The nascent carbon radical can be stabilized by a tritiated scavenger. This approach eliminates the use of heavy metal catalysts

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae along the Iguazu river basin

Directory of Open Access Journals (Sweden)

R. L. Lui

Full Text Available Abstract The Iguazu river is a tributary of the left margin of the Paraná river, isolated from this basin about 22 million years ago with the appearance of the Iguazu Falls. The Iguazu river is characterized by high endemism due to two factors: its rugged topography and the old isolation caused by formation of the Iguazu Falls. This study analyzed cytogenetically a population of Glanidium ribeiroi collected in a region at the final stretch of this basin, by Giemsa staining, C-banding, impregnation by silver nitrate, and FISH with probes of 5S rDNA, 18S rDNA, telomeric sequence [TTAGGG]n, and [GATA]n repeats. The diploid number was equal to 58 chromosomes. The heterochromatin was present in the terminal region of almost all chromosomes. The Ag-NORs were simple and presented interstitially on the short arm of the submetacentric pair 14, which was confirmed by FISH with 18S rDNA probe. The 5S rDNA-FISH marked only the submetacentric pair 16 on the long arm in interstitial position. The FISH with [TTAGGG]n probe presented all telomeres labeled as expected, with an absence of Interstitial Telomeric Sequence (ITS. The repetitive [GATA]n sequence was dispersed throughout the genome, with preferential location in the terminal region of all chromosomes. The data obtained are discussed herein with other species of Auchenipteridae, and other previously analyzed populations of G. ribeiroi from the Iguazu river, verifying differences among these populations, which should be mainly related to the rugged topography of this basin.

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats☆

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Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

2013-01-01

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. PMID:23648487

Hysteresis of magnetostructural transitions: Repeatable and non-repeatable processes

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Provenzano, Virgil; Della Torre, Edward; Bennett, Lawrence H.; ElBidweihy, Hatem

2014-02-01

The Gd5Ge2Si2 alloy and the off-stoichiometric Ni50Mn35In15 Heusler alloy belong to a special class of metallic materials that exhibit first-order magnetostructural transitions near room temperature. The magnetic properties of this class of materials have been extensively studied due to their interesting magnetic behavior and their potential for a number of technological applications such as refrigerants for near-room-temperature magnetic refrigeration. The thermally driven first-order transitions in these materials can be field-induced in the reverse order by applying a strong enough field. The field-induced transitions are typically accompanied by the presence of large magnetic hysteresis, the characteristics of which are a complicated function of temperature, field, and magneto-thermal history. In this study we show that the virgin curve, the major loop, and sequentially measured MH loops are the results of both repeatable and non-repeatable processes, in which the starting magnetostructural state, prior to the cycling of field, plays a major role. Using the Gd5Ge2Si2 and Ni50Mn35In15 alloys, as model materials, we show that a starting single phase state results in fully repeatable processes and large magnetic hysteresis, whereas a mixed phase starting state results in non-repeatable processes and smaller hysteresis.

Hysteresis of magnetostructural transitions: Repeatable and non-repeatable processes

International Nuclear Information System (INIS)

Provenzano, Virgil; Della Torre, Edward; Bennett, Lawrence H.; ElBidweihy, Hatem

2014-01-01

The Gd 5 Ge 2 Si 2 alloy and the off-stoichiometric Ni 50 Mn 35 In 15 Heusler alloy belong to a special class of metallic materials that exhibit first-order magnetostructural transitions near room temperature. The magnetic properties of this class of materials have been extensively studied due to their interesting magnetic behavior and their potential for a number of technological applications such as refrigerants for near-room-temperature magnetic refrigeration. The thermally driven first-order transitions in these materials can be field-induced in the reverse order by applying a strong enough field. The field-induced transitions are typically accompanied by the presence of large magnetic hysteresis, the characteristics of which are a complicated function of temperature, field, and magneto-thermal history. In this study we show that the virgin curve, the major loop, and sequentially measured MH loops are the results of both repeatable and non-repeatable processes, in which the starting magnetostructural state, prior to the cycling of field, plays a major role. Using the Gd 5 Ge 2 Si 2 and Ni 50 Mn 35 In 15 alloys, as model materials, we show that a starting single phase state results in fully repeatable processes and large magnetic hysteresis, whereas a mixed phase starting state results in non-repeatable processes and smaller hysteresis

AcmD, a homolog of the major autolysin AcmA of Lactococcus lactis, binds to the cell wall and contributes to cell separation and autolysis

NARCIS (Netherlands)

Visweswaran, Ganesh Ram R; Steen, Anton; Leenhouts, Kees; Szeliga, Monika; Ruban, Beata; Hesseling-Meinders, Anne; Dijkstra, Bauke W; Kuipers, Oscar P; Kok, Jan; Buist, Girbe

2013-01-01

Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under

Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation.

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Kanekura, Kohsuke; Yagi, Takuya; Cammack, Alexander J; Mahadevan, Jana; Kuroda, Masahiko; Harms, Matthew B; Miller, Timothy M; Urano, Fumihiko

2016-05-01

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the Chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This genetic alteration leads to the accumulation of five types of poly-dipeptides translated from the GGGGCC hexanucleotide repeat. Among these, poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) peptides are known to be neurotoxic. However, the mechanisms of neurotoxicity associated with these poly-dipeptides are not clear. A proteomics approach identified a number of interacting proteins with poly-PR peptide, including mRNA-binding proteins, ribosomal proteins, translation initiation factors and translation elongation factors. Immunostaining of brain sections from patients with C9orf72 ALS showed that poly-GR was colocalized with a mRNA-binding protein, hnRNPA1. In vitro translation assays showed that poly-PR and poly-GR peptides made insoluble complexes with mRNA, restrained the access of translation factors to mRNA, and blocked protein translation. Our results demonstrate that impaired protein translation mediated by poly-PR and poly-GR peptides plays a role in neurotoxicity and reveal that the pathways altered by the poly-dipeptides-mRNA complexes are potential therapeutic targets for treatment of C9orf72 FTD/ALS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

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Sadhukhan, Ratan; Chowdhury, Priyanka; Ghosh, Sourav; Ghosh, Utpal

2018-06-01

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

All-photonic quantum repeaters

Science.gov (United States)

Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

2015-01-01

Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories. PMID:25873153

Hydroxyapatite-binding peptides for bone growth and inhibition

Science.gov (United States)

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Reduction of starch granule size by expression of an engineered tandem starch-binding domain in potato plants

NARCIS (Netherlands)

Ji, Q.; Oomen, R.J.F.J.; Vincken, J.P.; Bolam, D.N.; Gilbert, H.J.; Suurs, L.C.J.M.; Visser, R.G.F.

2004-01-01

Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptice

Effects of dopaminergic drug treatments on in vivo radioligand binding to brain vesicular monoamine transporters

Energy Technology Data Exchange (ETDEWEB)

Kilbourn, Michael R; Frey, Kirk A; Vander Borght, Thierry; Sherman, Phillip S

1996-05-01

The effects of various dopaminergic drug treatments on the in vivo regional brain distribution of high-affinity radioligands ([{sup 11}C]dihydrotetrabenazine and [{sup 11}C]methoxytetrabenazine) for the rat brain vesicular monoamine transporter (VMAT2) were determined. Acute treatments with reserpine (2 mg/kg i.p.), tetrabenazine (10 mg/kg i.v.) or related benzoisoquinolines significantly reduced radiotracer binding in vivo. In contrast, radiotracer distributions remained unchanged after treatments with other dopaminergic drugs, whether given by single injection (haloperidol, 1 mg/kg i.p., pargyline 80 mg/kg), repeatedly (pargyline, 80 mg/kg s.c., 14 days), or by continuous infusion (deprenyl, 10 mg/kg/day, 5 days; L-DOPA methyl ester 100 mg/kg/day, 5 days). Repeated injections of tetrabenazine (5 mg/kg i.p., twice daily, 3 days) did not alter in vivo radioligand binding measured after allowing drug washout from the brain. These studies support the proposal that in vivo PET imaging of VMAT2 radioligands in patients with extrapyramidal movement disorders will not be affected by concurrent use of L-DOPA or deprenyl.

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

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Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

2010-12-29

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

Contingency blindness: location-identity binding mismatches obscure awareness of spatial contingencies and produce profound interference in visual working memory.

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Fiacconi, Chris M; Milliken, Bruce

2012-08-01

The purpose of the present study was to highlight the role of location-identity binding mismatches in obscuring explicit awareness of a strong contingency. In a spatial-priming procedure, we introduced a high likelihood of location-repeat trials. Experiments 1, 2a, and 2b demonstrated that participants' explicit awareness of this contingency was heavily influenced by the local match in location-identity bindings. In Experiment 3, we sought to determine why location-identity binding mismatches produce such low levels of contingency awareness. Our results suggest that binding mismatches can interfere substantially with visual-memory performance. We attribute the low levels of contingency awareness to participants' inability to remember the critical location-identity binding in the prime on a trial-to-trial basis. These results imply a close interplay between object files and visual working memory.

Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

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Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

2012-01-01

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

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Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

1992-04-01

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

Preliminary screening and identification of the hepatocarcinoma cell-binding peptide

International Nuclear Information System (INIS)

Zhu Xiaohua; Wu Hua

2004-01-01

Objective: To explore the feasibility of screening and isolating homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide library and to develop a new peptide which may be potentially used as targeting delivery carrier in the biological targeted diagnosis or therapy for liver cancer. Methods: A 12-mer peptide phage display library was used to screen and isolate peptides that bind to human hepatocarcinoma cells, and four rounds of subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones for human hepatocarcinoma cells were determined with enzyme-linked immunosorbent assay (ELISA) and compared with that to human liver cell and other tumor cells of different tissue origins, respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced through DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide, WH16, was determined with competitive inhibition test. Results: After four rounds of panning, the phages that were bound to and internalized in human hepatocarcinoma cells were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages for hepatocarcinoma cells. 56.67%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif . Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, that strongly support that cellular binding of the phage is mediated through its displayed peptide, and WH16 can also bind to HepG2. Conclusions: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide

Preliminary screening and identification of the hepatocarcinoma cell-binding peptide

Energy Technology Data Exchange (ETDEWEB)

Xiaohua, Zhu; Hua, Wu [Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong Univ. of Science and Technology, Wuhan (China)

2004-12-15

Objective: To explore the feasibility of screening and isolating homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide library and to develop a new peptide which may be potentially used as targeting delivery carrier in the biological targeted diagnosis or therapy for liver cancer. Methods: A 12-mer peptide phage display library was used to screen and isolate peptides that bind to human hepatocarcinoma cells, and four rounds of subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones for human hepatocarcinoma cells were determined with enzyme-linked immunosorbent assay (ELISA) and compared with that to human liver cell and other tumor cells of different tissue origins, respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced through DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide, WH16, was determined with competitive inhibition test. Results: After four rounds of panning, the phages that were bound to and internalized in human hepatocarcinoma cells were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages for hepatocarcinoma cells. 56.67%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif . Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, that strongly support that cellular binding of the phage is mediated through its displayed peptide, and WH16 can also bind to HepG2. Conclusions: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display random peptide

Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

Science.gov (United States)

Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

2011-01-01

MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

Science.gov (United States)

Wohleb, Eric S; Hanke, Mark L; Corona, Angela W; Powell, Nicole D; Stiner, La'Tonia M; Bailey, Michael T; Nelson, Randy J; Godbout, Jonathan P; Sheridan, John F

2011-04-27

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

78 FR 65594 - Vehicular Repeaters

Science.gov (United States)

2013-11-01

... coordinators estimate the effect on coordination fees? Does the supposed benefit that mobile repeater stations... allow the licensing and operation of vehicular repeater systems and other mobile repeaters by public... email: [email protected] or phone: 202-418- 0530 or TTY: 202-418-0432. For detailed instructions for...

Tevatron serial data repeater system

International Nuclear Information System (INIS)

Ducar, R.J.

1981-01-01

A ten megabit per second serial data repeater system has been developed for the 6.28km Tevatron accelerator. The repeaters are positioned at each of the thirty service buildings and accommodate control and abort system communications as well as distribution of the Tevatron time and energy clocks. The repeaters are transparent to the particular protocol of the transmissions. Serial data are encoded locally as unipolar two volt signals employing the self-clocking Manchester Bi-Phase code. The repeaters modulate the local signals to low-power bursts of 50 MHz rf carrier for the 260m transmission between service buildings. The repeaters also demodulate the transmission and restructure the data for local utilization. The employment of frequency discrimination techniques yields high immunity to the characteristic noise spectrum

Salicylic-Acid-Induced Chilling- and Oxidative-Stress Tolerance in Relation to Gibberellin Homeostasis, C-Repeat/Dehydration-Responsive Element Binding Factor Pathway, and Antioxidant Enzyme Systems in Cold-Stored Tomato Fruit.

Science.gov (United States)

Ding, Yang; Zhao, Jinhong; Nie, Ying; Fan, Bei; Wu, Shujuan; Zhang, Yu; Sheng, Jiping; Shen, Lin; Zhao, Ruirui; Tang, Xuanming

2016-11-02

Effects of salicylic acid (SA) on gibberellin (GA) homeostasis, C-repeat/dehydration-responsive element binding factor (CBF) pathway, and antioxidant enzyme systems linked to chilling- and oxidative-stress tolerance in tomato fruit were investigated. Mature green tomatoes (Solanum lycopersicum L. cv. Moneymaker) were treated with 0, 0.5, and 1 mM SA solution for 15 min before storage at 4 °C for 28 days. In comparison to 0 or 0.5 mM SA, 1 mM SA significantly decreased the chilling injury (CI) index in tomato fruit. In the SA-treated fruit, the upregulation of GA biosynthetic gene (GA3ox1) expression was followed by gibberellic acid (GA 3 ) surge and DELLA protein degradation. CBF1 participated in the SA-modulated tolerance and stimulated the expression of GA catabolic gene (GA2ox1). Furthermore, 1 mM SA enhanced activities of antioxidant enzymes and, thus, reduced reactive oxygen species accumulation. Our findings suggest that SA might protect tomato fruit from CI and oxidative damage through regulating GA metabolism, CBF1 gene expression, and antioxidant enzyme activities.

Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation▿

Science.gov (United States)

Sanders, Steven L.; Arida, Ahmad R.; Phan, Funita P.

2010-01-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability. PMID:20679488

Requirement for the phospho-H2AX binding module of Crb2 in double-strand break targeting and checkpoint activation.

Science.gov (United States)

Sanders, Steven L; Arida, Ahmad R; Phan, Funita P

2010-10-01

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

Science.gov (United States)

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-08-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Repeat Customer Success in Extension

Science.gov (United States)

Bess, Melissa M.; Traub, Sarah M.

2013-01-01

Four multi-session research-based programs were offered by two Extension specialist in one rural Missouri county. Eleven participants who came to multiple Extension programs could be called "repeat customers." Based on the total number of participants for all four programs, 25% could be deemed as repeat customers. Repeat customers had…

Film repeats in radiology department

International Nuclear Information System (INIS)

Suwan, A. Z.; Al-Shakharah, A. I

1997-01-01

During a one year period, 4910 radiographs of 55780 films were repeated. The objective of our study was to analyse and to classify the causes in order to minimize the repeats, cut the expenses and to provide optimal radiographs for accurate diagnosis. Analysis of the different factors revealed that, 43.6% of film repeats in our service were due to faults in exposure factors, centering comprises 15.9% of the repeats, while too much collimation was responsible for 7.6% of these repeats. All of which can be decreased by awareness and programmed training of technicians. Film blurring caused by patient motion was also responsible for 4.9% for radiographs reexamination, which can be minimized by detailed explanation to the patient and providing the necessary privacy. Fogging of X-Ray films by improper storage or inadequate handling or processing faults were responsible for 14.5% in repeats in our study. Methods and criteria for proper storage and handling of films were discussed. Recommendation for using modern day-light and laser processor has been high lighted. Artefacts are noticeably high in our cases, due to spinal dresses and frequent usage of precious metals for c osmotic purposes in this part of the world. The repeated films comprise 8.8% of all films We conclude that, the main factor responsible for repeats of up to 81.6% of cases was the technologists, thus emphasizing the importance of adequate training of the technologists. (authors). 15 refs., 9 figs., 1 table

Repeat migration and disappointment.

Science.gov (United States)

Grant, E K; Vanderkamp, J

1986-01-01

This article investigates the determinants of repeat migration among the 44 regions of Canada, using information from a large micro-database which spans the period 1968 to 1971. The explanation of repeat migration probabilities is a difficult task, and this attempt is only partly successful. May of the explanatory variables are not significant, and the overall explanatory power of the equations is not high. In the area of personal characteristics, the variables related to age, sex, and marital status are generally significant and with expected signs. The distance variable has a strongly positive effect on onward move probabilities. Variables related to prior migration experience have an important impact that differs between return and onward probabilities. In particular, the occurrence of prior moves has a striking effect on the probability of onward migration. The variable representing disappointment, or relative success of the initial move, plays a significant role in explaining repeat migration probabilities. The disappointment variable represents the ratio of actural versus expected wage income in the year after the initial move, and its effect on both repeat migration probabilities is always negative and almost always highly significant. The repeat probabilities diminish after a year's stay in the destination region, but disappointment in the most recent year still has a bearing on the delayed repeat probabilities. While the quantitative impact of the disappointment variable is not large, it is difficult to draw comparisons since similar estimates are not available elsewhere.

NEIL3 Repairs Telomere Damage during S Phase to Secure Chromosome Segregation at Mitosis

Directory of Open Access Journals (Sweden)

Jia Zhou

2017-08-01

Full Text Available Oxidative damage to telomere DNA compromises telomere integrity. We recently reported that the DNA glycosylase NEIL3 preferentially repairs oxidative lesions in telomere sequences in vitro. Here, we show that loss of NEIL3 causes anaphase DNA bridging because of telomere dysfunction. NEIL3 expression increases during S phase and reaches maximal levels in late S/G2. NEIL3 co-localizes with TRF2 and associates with telomeres during S phase, and this association increases upon oxidative stress. Mechanistic studies reveal that NEIL3 binds to single-stranded DNA via its intrinsically disordered C terminus in a telomere-sequence-independent manner. Moreover, NEIL3 is recruited to telomeres through its interaction with TRF1, and this interaction enhances the enzymatic activity of purified NEIL3. Finally, we show that NEIL3 interacts with AP Endonuclease 1 (APE1 and the long-patch base excision repair proteins PCNA and FEN1. Taken together, we propose that NEIL3 protects genome stability through targeted repair of oxidative damage in telomeres during S/G2 phase.

Repeated prenatal exposure to valproic acid results in cerebellar hypoplasia and ataxia.

Science.gov (United States)

Main, Stacey L; Kulesza, Randy J

2017-01-06

Autism spectrum disorder (ASD) is a developmental brain disorder characterized by restricted and repetitive patterns of behavior, social and communication defects, and is commonly associated with difficulties with motor coordination. The etiology of ASD, while mostly idiopathic, has been linked to hereditary factors and teratogens, such as valproic acid (VPA). VPA is used clinically to treat epilepsy, mood disorders, and in the prevention of migraines. The use of VPA during pregnancy significantly increases the risk of ASD in the offspring. Neuropathological studies show decreased cerebellar function in patients with ASD, resulting in gait, balance and coordination impairments. Herein, we have exposed pregnant rats to a repeated oral dose of VPA on embryonic days 10 and 12 and performed a detailed investigation of the structure and function of the cerebellar vermis. We found that throughout all ten lobules of the cerebellar vermis, Purkinje cells were significantly smaller and expression of the calcium binding protein calbindin (CB) was significantly reduced. We also found that dendritic arbors of Purkinje cells were shorter and less complex. Additionally, animals exposed to a repeated dose of VPA performed significantly worse in a number of motor tasks, including beam walking and the rotarod. These results suggest that repeated embryonic exposure to VPA induces significant cerebellar dysfunction and is an effective animal model to study the cerebellar alterations in ASD. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Quantum repeated games revisited

International Nuclear Information System (INIS)

Frąckiewicz, Piotr

2012-01-01

We present a scheme for playing quantum repeated 2 × 2 games based on Marinatto and Weber’s approach to quantum games. As a potential application, we study the twice repeated Prisoner’s Dilemma game. We show that results not available in the classical game can be obtained when the game is played in the quantum way. Before we present our idea, we comment on the previous scheme of playing quantum repeated games proposed by Iqbal and Toor. We point out the drawbacks that make their results unacceptable. (paper)

FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription

International Nuclear Information System (INIS)

Holmqvist, Per-Henrik; Belikov, Sergey; Zaret, Kenneth S.; Wrange, Oerjan

2005-01-01

Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription. The proximal FoxA binding segment consisted of two individual FoxA sites at -57/-46 and -45/-34, respectively, that mediated an increased basal MMTV transcription. FoxA1 binding altered the chromatin structure of both the inactive- and the hormone-activated MMTV LTR. Hydroxyl radical foot printing revealed FoxA1-mediated changes in the nucleosome arrangement. Micrococcal nuclease digestion showed the hormone-dependent sub-nucleosome complex, containing ∼120 bp of DNA, to be expanded by FoxA1 binding to the proximal segment into a larger complex containing ∼200 bp. The potential function of the FoxA1-mediated expression of the MMTV provirus for maintenance of expression in different tissues is discussed

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers.

Science.gov (United States)

Xu, Jing-Yuan; Zhu, Yan; Yi, Ze; Wu, Gang; Xie, Guo-Yong; Qin, Min-Jian

2018-01-01

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum. Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Toward the design of new DNA G-quadruplex ligands through rational analysis of polymorphism and binding data.

Science.gov (United States)

Artese, Anna; Costa, Giosuè; Distinto, Simona; Moraca, Federica; Ortuso, Francesco; Parrotta, Lucia; Alcaro, Stefano

2013-10-01

Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Science.gov (United States)

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Presence of fibronectin-binding protein gene prtF2 in invasive group A streptococci in tropical Australia is associated with increased internalisation efficiency.

Science.gov (United States)

Gorton, Davina; Norton, Robert; Layton, Ramon; Smith, Helen; Ketheesan, Natkunam

2005-03-01

The fibronectin-binding proteins (FnBPs) PrtF1 and PrtF2 are considered to be major group A streptococcal virulence factors, mediating adherence to and internalisation of host cells. The present study investigated an association between the presence of prtF1 and prtF2 genes and internalisation efficiency in group A streptococci (GAS) isolated from patients with invasive disease. Of the 80 isolates tested, 58 (73%) had prtF1 and 71 (89%) possessed prtF2. Three isolates (4%) had neither gene, seven (9%) had prtF1 only, 19 (24%) had prtF2 only and 51 isolates (64%) had both prtF1 and prtF2. prtF2-positive isolates internalised up to three times more efficiently than isolates that had prtF1 alone (Pinternalisation efficiency and presence of the prtF1 gene. Analysis of the fibronectin-binding repeat domain (FBRD) of prtF2 revealed that this gene can contain 2, 3, 4 or 5 repeat regions and that five repeat regions conferred very high internalisation efficiency in invasive GAS isolates.

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting

Science.gov (United States)

Yuan, Qi; Eckland, Thomas; Telling, Glenn; Bartz, Jason; Bartelt-Hunt, Shannon

2015-01-01

Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrPSc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrPSc or consolidation of the bonding between PrPSc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions. PMID:25665187

Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats.

Science.gov (United States)

Panigrahi, Gagan B; Slean, Meghan M; Simard, Jodie P; Pearson, Christopher E

2012-12-07

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

Repeatability of visual acuity measurement.

Science.gov (United States)

Raasch, T W; Bailey, I L; Bullimore, M A

1998-05-01

This study investigates features of visual acuity chart design and acuity testing scoring methods which affect the validity and repeatability of visual acuity measurements. Visual acuity was measured using the Sloan and British Standard letter series, and Landolt rings. Identifiability of the different letters as a function of size was estimated, and expressed in the form of frequency-of-seeing curves. These functions were then used to simulate acuity measurements with a variety of chart designs and scoring criteria. Systematic relationships exist between chart design parameters and acuity score, and acuity score repeatability. In particular, an important feature of a chart, that largely determines the repeatability of visual acuity measurement, is the amount of size change attributed to each letter. The methods used to score visual acuity performance also affect repeatability. It is possible to evaluate acuity score validity and repeatability using the statistical principles discussed here.

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

International Nuclear Information System (INIS)

Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

2009-01-01

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3ζ. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3ζ is ∼3-folds higher than that between unphosphorylated 4R-tau and 14-3-3ζ. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3ζ to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3ζ. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3ζ exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3ζ suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

Science.gov (United States)

Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

2010-11-18

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

Science.gov (United States)

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Preliminary screening and identification of the peptide binding to hepatocarcinoma cell

International Nuclear Information System (INIS)

Zhu Xiaohua; Wu Ha

2004-01-01

Objective: The present study was performed to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide library with the purpose of developing a new peptide which may be potentially used as target delivery carrier in the biological target diagnosis or therapy for liver cancer. Methods: A peptide 12-mer phage display library was used to screen and isolate peptide that bind to human hepatocarcinoma cell, and four rounds subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones to human hepatocarcinoma cell were determined with ELISA and compared with human liver cell and other tumor cells of different tissue origins respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced though DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide WH16 was determined with competitive inhibition test. Results: After four rounds panning, the phages that bound to and internalized in human hepatocarcinoma cell were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages to hepatpcarcinoma cells 56.57%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif. Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, which strongly support that cellular binding of phage is mediated though its displayed peptide and WH16 can also bind to HepG2. Conclusion: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide libraries. The sequence of peptide that can bind to

Preliminary screening and identification of the peptide binding to hepatocarcinoma cell

Energy Technology Data Exchange (ETDEWEB)

Xiaohua, Zhu; Ha, Wu [Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China)

2004-07-01

Objective: The present study was performed to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide library with the purpose of developing a new peptide which may be potentially used as target delivery carrier in the biological target diagnosis or therapy for liver cancer. Methods: A peptide 12-mer phage display library was used to screen and isolate peptide that bind to human hepatocarcinoma cell, and four rounds subtractive panning were carried out with the human hepatocarcinoma cell line HepG2 as the target. The affinities of selected phage clones to human hepatocarcinoma cell were determined with ELISA and compared with human liver cell and other tumor cells of different tissue origins respectively. In addition, the binding site in the tumor cells was observed with immunofluorescence analysis under confocal light microscopy. The amino acid sequences of phages that bind HepG2 specifically were deduced though DNA sequencing. Based on the results of DNA sequence, a 16-mer peptide (WH16) was designed and synthesized. Binding ability of the new peptide WH16 was determined with competitive inhibition test. Results: After four rounds panning, the phages that bound to and internalized in human hepatocarcinoma cell were isolated. ELISA and immunofluorescence analysis confirmed the affinity of these phages to hepatpcarcinoma cells 56.57%(17/30) of the isolated phages displayed repeated sequence FLLEPHLMDTSM, and FLEP was defined as conservative motif. Binding of the selected phage to HepG2 cells was inhibited by synthesized peptide WH16, which strongly support that cellular binding of phage is mediated though its displayed peptide and WH16 can also bind to HepG2. Conclusion: It is feasible to screen and isolate homing peptides that bind specifically, or preferentially, to hepatocarcinoma cells using phage display of random peptide libraries. The sequence of peptide that can bind to

T-cell mean telomere lengths changes in treatment naïve HIV-infected patients randomized to G-CSF or placebo simultaneously with initiation of HAART

DEFF Research Database (Denmark)

Aladdin, H; Von Essen, M; Schjerling, P

2001-01-01

The effect of highly active antiretroviral therapy (HAART) and granulocyte colony stimulating factor (G-CSF) on mean telomere restriction fragment (TRF) length of peripheral blood mononuclear cells (PBMC) was examined in 11 treatment naïve human immunodeficiency virus (HIV)-infected individuals...... with a CD4+ T-cell count CSF thrice weekly for 12 weeks (n = 6) or placebo (n = 5). An increase in the mean TRF lengths was observed in PBMC of patients on HAART after 24 weeks of treatment mainly owing to increased mean CD8+ T-cell TRF...... lengths. However, in the group of patients on HAART combined with G-CSF no changes of PBMC mean TRF length was observed during treatment or during 12 weeks of follow-up. The mean CD4+ T-cell TRF length did not change in any of the two groups. These results confirm that HAART induces mainly the lengthening...

Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

Science.gov (United States)

Amirhaeri, S; Wohlrab, F; Wells, R D

1995-02-17

The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig proteins.

Directory of Open Access Journals (Sweden)

Rajeev Raman

Full Text Available BACKGROUND: Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. PRINCIPAL FINDINGS: We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th (Lig A9 and 10(th repeats (Lig A10; and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon. All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm, probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. CONCLUSIONS: We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

Binding of fluorine-18 by the oral bacterium, Streptococcus mutans

Energy Technology Data Exchange (ETDEWEB)

Yotis, W.W.; Mante, S.; Brennan, P.C.; Kirchner, F.R.; Glendenin, L.E.

1979-01-01

The binding of carrier-free fluorine-18 by resting cells of the cariogenic microorganism Streptococcus mutans GS-5 was assessed. A Ge(Li)..gamma..-ray spectrometer attached to a 4096 channel pulse-height analyzer was used to measure the /sup 18/F bound and to check the radiochemical purity of /sup 18/F. The binding was dependent on time, pH, the amount of /sup 18/F used, the cell status and the fluoride concentration. The adherence of /sup 18/F to Strep. mutans did not require addition of an exogenous energy source, such as glucose, and proceeded equally well at 4 to 37/sup 0/C or at varying oxygen tensions. Under optimal conditions, resting cells of the strain bound approximately 10/sup 9/ atoms of /sup 18/F and more than 10/sup 13/ atoms of total fluoride in the presence of 10 parts/10/sup 6/ NaF per mg dry weight of cells that were not removed by repeated washings.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

Science.gov (United States)

Rangl, Martina; Leitner, Michael; Riihimäki, Tiina; Lehtonen, Soili; Hytönen, Vesa P; Gruber, Hermann J; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

2014-02-01

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(β)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(β) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.

The last half-repeat of transcription activator-like effector (TALE) is dispensable and thereby TALE-based technology can be simplified.

Science.gov (United States)

Zheng, Chong-Ke; Wang, Chun-Lian; Zhang, Xiao-Ping; Wang, Fu-Jun; Qin, Teng-Fei; Zhao, Kai-Jun

2014-09-01

To activate the expression of host genes that contribute to pathogen growth, pathogenic Xanthomonas bacteria inject their transcription activator-like effectors (TALEs) into plant cells and the TALEs bind to target gene promoters by the central repeat region consisting of near-perfect 34-amino-acid repeats (34-aa repeats). Based on the recognition codes between the 34-aa repeats and the targeted nucleotides, TALE-based technologies, such as designer TALEs (dTALEs) and TALE nucleases (TALENs), have been developed. Amazingly, every natural TALE invariantly has a truncated last half-repeat (LHR) at the end of the 34-aa repeats. Consequently, all the reported dTALEs and TALENs also harbour their LHRs. Here, we show that the LHRs in dTALEs are dispensable for the function of gene activation by both transient expression assays in Nicotiana benthamiana and gene-specific targeting in the rice genome, indicating that TALEs might originate from a single progenitor. In the light of this finding, we demonstrate that dTALEs can be constructed through two simple steps. Moreover, the activation strengths of dTALEs lacking the LHR are comparable with those of dTALEs harbouring the LHR. Our results provide new insights into the origin of natural TALEs, and will facilitate the simplification of the design and assembly of TALE-based tools, such as dTALEs and TALENs, in the near future. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Fostering repeat donations in Ghana.

Science.gov (United States)

Owusu-Ofori, S; Asenso-Mensah, K; Boateng, P; Sarkodie, F; Allain, J-P

2010-01-01

Most African countries are challenged in recruiting and retaining voluntary blood donors by cost and other complexities and in establishing and implementing national blood policies. The availability of replacement donors who are a cheaper source of blood has not enhanced repeat voluntary donor initiatives. An overview of activities for recruiting and retaining voluntary blood donors was carried out. Donor records from mobile sessions were reviewed from 2002 to 2008. A total of 71,701 blood donations; 45,515 (63.5%) being voluntary donations with 11,680 (25%) repeat donations were collected during the study period. Donations from schools and colleges contributed a steady 60% of total voluntary whilst radio station blood drives increased contribution from 10 to 27%. Though Muslim population is less than 20%, blood collection was above the 30-donation cost-effectiveness threshold with a repeat donation trend reaching 60%. In contrast Christian worshippers provided donations. Repeat donation trends amongst school donors and radio blood drives were 20% and 70% respectively. Repeat donations rates have been variable amongst different blood donor groups in Kumasi, Ghana. The impact of community leaders in propagating altruism cannot be overemphasized. Programs aiming at motivating replacement donors to be repeat donors should be developed and assessed. Copyright 2009 The International Association for Biologicals. All rights reserved.

A simple repeat polymorphism in the MITF-M promoter is a key regulator of white spotting in dogs.

Directory of Open Access Journals (Sweden)

Izabella Baranowska Körberg

Full Text Available The white spotting locus (S in dogs is colocalized with the MITF (microphtalmia-associated transcription factor gene. The phenotypic effects of the four S alleles range from solid colour (S to extreme white spotting (s(w. We have investigated four candidate mutations associated with the s(w allele, a SINE insertion, a SNP at a conserved site and a simple repeat polymorphism all associated with the MITF-M promoter as well as a 12 base pair deletion in exon 1B. The variants associated with white spotting at all four loci were also found among wolves and we conclude that none of these could be a sole causal mutation, at least not for extreme white spotting. We propose that the three canine white spotting alleles are not caused by three independent mutations but represent haplotype effects due to different combinations of causal polymorphisms. The simple repeat polymorphism showed extensive diversity both in dogs and wolves, and allele-sharing was common between wolves and white spotted dogs but was non-existent between solid and spotted dogs as well as between wolves and solid dogs. This finding was unexpected as Solid is assumed to be the wild-type allele. The data indicate that the simple repeat polymorphism has been a target for selection during dog domestication and breed formation. We also evaluated the significance of the three MITF-M associated polymorphisms with a Luciferase assay, and found conclusive evidence that the simple repeat polymorphism affects promoter activity. Three alleles associated with white spotting gave consistently lower promoter activity compared with the allele associated with solid colour. We propose that the simple repeat polymorphism affects cooperativity between transcription factors binding on either flanking sides of the repeat. Thus, both genetic and functional evidence show that the simple repeat polymorphism is a key regulator of white spotting in dogs.

Effect of repeat unit structure and molecular mass of lactic acid bacteria hetero-exopolysaccharides on binding to milk proteins

DEFF Research Database (Denmark)

Birch, Johnny; HarÐarson, HörÐur Kári; Khan, Sanaullah

2017-01-01

-exopolysaccharides (HePSs) of 0.14–4.9 MDa from lactic acid bacteria to different milk proteins (β-casein, κ-casein, native and heat-treated β-lactoglobulin) at pH 4.0–5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold......, respectively, in the complexation with native β-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated β-lactoglobulin and β-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic......Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero...

Repeated DNA sequences in fungi

Energy Technology Data Exchange (ETDEWEB)

Dutta, S K

1974-11-01

Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10 to 20 percent repeated DNA sequences. There are approximately 100 to 110 copies of repeated DNA sequences of approximately 4 x 10/sup 7/ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5/sup 0/C difference of T/sub e/50 (temperature at which 50 percent duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA. (auth)

Effects of saw palmetto extract on micturition reflex of rats and its autonomic receptor binding activity.

Science.gov (United States)

Oki, Tomomi; Suzuki, Mayumi; Nishioka, Yasuhiko; Yasuda, Akio; Umegaki, Keizo; Yamada, Shizuo

2005-04-01

We examined the effects of saw palmetto extract (SPE) on the rat micturition reflex and on autonomic receptors in the lower urinary tract. The effect of SPE was examined on cystometrograms of anesthetized rats induced by intravesical infusion of saline or 0.1% acetic acid. SHR/NDmc-cp (cp/cp) rats received repeat oral administration of SPE and nighttime urodynamic function was determined. The autonomic receptor binding activity of SPE in the rat bladder and prostate was examined by radioligand binding assay. Intraduodenal administration of SPE (60 mg/kg) in anesthetized rat cystometry caused a significant increase in the micturition interval, micturition volume and bladder capacity during intravesical saline infusion. Also, similar administration of SPE at doses of 12 and 20 mg/kg significantly reversed the shortened micturition interval as well as the decreased micturition volume and bladder capacity due to 0.1% acetic acid infusion in a dose dependent manner. In conscious SHR/NDmc-cp (cp/cp) rats repeat oral administration of SPE (6 mg/kg daily) constantly increased the micturition interval and concomitantly decreased voiding frequency. SPE inhibited specific binding of [H]NMS ([N-methyl-H]scopolamine methyl chloride) (bladder) and [H]prazosin (prostate) with IC50 values of 46.1 and 183 microg/ml, respectively. SPE significantly alleviates urodynamic symptoms in hyperactive rat bladders by increasing bladder capacity and subsequently prolonging the micturition interval. Our data may support the clinical efficacy of SPE for the treatment of lower urinary tract symptoms.

Topological characteristics of helical repeat proteins

NARCIS (Netherlands)

Groves, M R; Barford, D

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem

The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

NARCIS (Netherlands)

Fenyk, S.; Townsend, P.D.; Dixon, C.H.; Spies, G.B.; Campillo, A.S.E.; Slootweg, E.J.; Westerhof, L.B.; Gawehns, F.K.K.; Knight, M.R.; Sharples, G.J.; Goverse, A.; Palsson, L.O.; Takken, F.L.W.; Cann, M.J.

2015-01-01

Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1

Repeated administration of alpha7 nicotinic acetylcholine receptor (nAChR) agonists, but not positive allosteric modulators, increases alpha7 nAChR levels in the brain

DEFF Research Database (Denmark)

Christensen, Ditte Z; Mikkelsen, Jens D; Hansen, Henrik H

2010-01-01

AChR binding sites in several brain regions, particularly in the prefrontal cortex. The alpha7 nAChR agonists SSR180711 and PNU-282987 also increase [(125)I]-BTX binding, suggesting that this is a general consequence of alpha7 nAChR agonism. Interestingly, the alpha7 nAChR positive allosteric modulators PNU......The alpha7 nicotinic acetylcholine receptor (nAChR) is an important target for treatment of cognitive deficits in schizophrenia and Alzheimer's disease. However, the receptor desensitizes rapidly in vitro, which has led to concern regarding its applicability as a clinically relevant drug target....... Here we investigate the effects of repeated agonism on alpha7 nAChR receptor levels and responsiveness in vivo in rats. Using [(125)I]-alpha-bungarotoxin (BTX) autoradiography we show that acute or repeated administration with the selective alpha7 nAChR agonist A-582941 increases the number of alpha7 n...

Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities.

Science.gov (United States)

Blackwood, Christopher B; Hudleston, Deborah; Zak, Donald R; Buyer, Jeffrey S

2007-08-01

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Science.gov (United States)

Pelham, Christopher; Jimenez, Tamara; Rodova, Marianna; Rudolph, Angela; Chipps, Elizabeth; Islam, M Rafiq

2013-12-01

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression. © 2013.

The triplet repeats of the Sin Nombre hantavirus 5' untranslated region are sufficient in cis for nucleocapsid-mediated translation initiation.

Science.gov (United States)

Mir, Mohammad A; Panganiban, Antonito T

2010-09-01

Hantavirus nucleocapsid protein (N) can replace the cellular cap-binding complex, eukaryotic initiation factor 4F (eIF4F), to mediate translation initiation. Although N can augment translation initiation of nonviral mRNA, initiation of viral mRNA by N is superior. All members of the Bunyaviridae family, including the species of the hantavirus genus, express either three or four primary mRNAs from their tripartite negative-sense genomes. The 5' ends of the mRNAs contain nonviral heterologous oligonucleotides that originate from endonucleolytic cleavage of cellular mRNA during the process of cap snatching. In the hantaviruses these caps terminate with a 3' G residue followed by nucleotides arising from the viral template. Further, the 5' untranslated region (UTR) of viral mRNA uniformly contains, near the 5' end, either two or three copies of the triplet repeat sequence, UAGUAG or UAGUAGUAG. Through analysis of a panel of mutants with mutations in the viral UTR, we found that the sequence GUAGUAG is sufficient for preferential N-mediated translation initiation and for high-affinity binding of N to the UTR. This heptanucleotide sequence is present in viral mRNA containing either two or three copies of the triplet repeat.

Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

Directory of Open Access Journals (Sweden)

V Chandana Epa

Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).

Science.gov (United States)

Xu, Jin-Gen; Huang, Chunfeng; Yang, Zhengfeng; Jin, Mengmeng; Fu, Panhan; Zhang, Ni; Luo, Jian; Li, Dali; Liu, Mingyao; Zhou, Yan; Zhu, Yongqun

2015-01-23

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Energy Technology Data Exchange (ETDEWEB)

Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas; Patel, Dinshaw J.

2015-09-08

RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.

UK 2009-2010 repeat station report

Directory of Open Access Journals (Sweden)

Thomas J.G. Shanahan

2013-03-01

Full Text Available The British Geological Survey is responsible for conducting the UK geomagnetic repeat station programme. Measurements made at the UK repeat station sites are used in conjunction with the three UK magnetic observatories: Hartland, Eskdalemuir and Lerwick, to produce a regional model of the local field each year. The UK network of repeat stations comprises 41 stations which are occupied at approximately 3-4 year intervals. Practices for conducting repeat station measurements continue to evolve as advances are made in survey instrumentation and as the usage of the data continues to change. Here, a summary of the 2009 and 2010 UK repeat station surveys is presented, highlighting the measurement process and techniques, density of network, reduction process and recent results.

Complexities in human herpesvirus-6A and -6B binding to host cells

International Nuclear Information System (INIS)

Pedersen, Simon Metz; Hoellsberg, Per

2006-01-01

Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher affinity than others for the virus. Within recent years, elucidation of the viral complex has identified additional HHV-6A and -6B specific glycoproteins. Thus, gH-gL associates with a gQ1-gQ2 dimer to form a heterotetrameric complex. In addition, a novel complex consisting of gH-gL-gO has been described that does not bind CD46. Accumulating evidence suggests that an additional HHV-6A and -6B receptor exists. The previous simple picture of HHV-6A/B-host cell contact therefore includes more layers of complexities on both the viral and the host cell side of the interaction

Reject/repeat analysis and the effect prior film viewing has on a department's reject/repeat rate

International Nuclear Information System (INIS)

Clark, P.A.; Hogg, P.

2003-01-01

Purpose: Achieving cost-effectiveness within the NHS is an old initiative but one that has again been highlighted by recent government policies (The New NHS-Modern and Dependable, Stationary Office, London, 1997). It has been reiterated that it is the responsibility of individual Trusts to devise means to provide such a service. Reject/repeat analyses have long been the primary tool used to assess the cost-effectiveness of radiography departments (Quality Assurance in Diagnostic Radiology, WHO, Geneva, 1982). This research paper examines an in-house initiative (viewing patients' previous films) commonly employed in other Health Trusts in order to reduce departmental repeat/reject rates. Method: Three hundred orthopaedic patients with hip, knee and ankle prostheses were included in a reject/repeat analysis. The aim was to investigate whether or not viewing patient's previous relevant radiographs would be advantageous to the practicing radiographer. This was done through an audit cycle consisting of two audit periods each lasting for 3 months. The primary audit period recorded the baseline repeat/reject rate, with the secondary audit period recording the repeat/reject rate under an experimental condition of viewing the relevant radiographs. Results: The baseline audit revealed repeat rates of 33% in orthopaedic patients with hip, knee and ankle prostheses. The availability of prior film viewing to the radiographer reduced this repeat rate to 10.6%. Conclusion: Prior film viewing dramatically reduced the department's repeat/reject rate by 22.4%. This provides scope for significant patient dose reductions as well as reducing departmental film expenses. This is an underestimated initiative and should be used appropriately in routine clinical practice

Neural correlates of binding lyrics and melodies for the encoding of new songs.

Science.gov (United States)

Alonso, Irene; Davachi, Lila; Valabrègue, Romain; Lambrecq, Virginie; Dupont, Sophie; Samson, Séverine

2016-02-15

Songs naturally bind lyrics and melody into a unified representation. Using a subsequent memory paradigm, we examined the neural processes associated with binding lyrics and melodies during song encoding. Participants were presented with songs in two conditions: a unified condition (melodies sung with lyrics), and a separate condition (melodies sung with the syllable "la"). In both cases, written lyrics were displayed and participants were instructed to memorize them by repeating them covertly or by generating mental images of the songs. We expected the unified condition to recruit the posterior superior temporal gyrus, known to be involved in perceptual integration of songs, as well as the left inferior frontal gyrus (IFG). Conversely, we hypothesized that the separate condition would engage a larger network including the hippocampus to bind lyrics and melodies of songs, and the basal ganglia and the cerebellum to ensure the correct sequence coupling of verbal and musical information in time. Binding lyrics and melodies in the unified condition revealed activation of the left IFG, bilateral middle temporal gyrus (MTG), and left motor cortex, suggesting a strong linguistic processing for this condition. Binding in the separate compared to the unified condition revealed greater activity in the right hippocampus as well as other areas including the left caudate, left cerebellum, and right IFG. This study provides novel evidence for the role of the right hippocampus in binding lyrics and melodies in songs. Results are discussed in light of studies of binding in the visual domain and highlight the role of regions involved in timing and synchronization such as the basal ganglia and the cerebellum. Copyright © 2015. Published by Elsevier Inc.

Application of pressure ultrafiltration in determining the binding capacity of drugs to human albumin and to plasma proteins of intact and irradiated rat females

International Nuclear Information System (INIS)

Zima, M.

1976-01-01

The significance of the binding of drugs to plasma proteins has repeatedly been demonstrated and draws the interest of many pharmacologists. The described experiments served to study the binding of isoniazid (INH) to human albumin of various dilution and to whole plasma proteins of irradiated (on the Oth, 3rd and 6th day after exposure to 154.8 mC/kg=600 R) and non-irradiated rats using the technique of modified accelerated ultrafiltration through cellophane. The total characteristics of the binding and its changes were demonstrated by the equilibrium constant, the numbers of binding sites and the changes of free binding energy. The results show that the dilution of human albumin affects the strength of the INH binding on this albumin and further that the normally weak INH binding is diminished even more in irradiated rats. This cannot be explained by the change in the percentage composition of the rat plasma. (author)

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

Directory of Open Access Journals (Sweden)

Charles Sabin

Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

A specific family of interspersed repeats (SINEs facilitates meiotic synapsis in mammals

Directory of Open Access Journals (Sweden)

Johnson Matthew E

2013-01-01

Full Text Available Abstract Background Errors during meiosis that affect synapsis and recombination between homologous chromosomes contribute to aneuploidy and infertility in humans. Despite the clinical relevance of these defects, we know very little about the mechanisms by which homologous chromosomes interact with one another during mammalian meiotic prophase. Further, we remain ignorant of the way in which chromosomal DNA complexes with the meiosis-specific structure that tethers homologs, the synaptonemal complex (SC, and whether specific DNA elements are necessary for this interaction. Results In the present study we utilized chromatin immunoprecipitation (ChIP and DNA sequencing to demonstrate that the axial elements of the mammalian SC are markedly enriched for a specific family of interspersed repeats, short interspersed elements (SINEs. Further, we refine the role of the repeats to specific sub-families of SINEs, B1 in mouse and AluY in old world monkey (Macaca mulatta. Conclusions Because B1 and AluY elements are the most actively retrotransposing SINEs in mice and rhesus monkeys, respectively, our observations imply that they may serve a dual function in axial element binding; i.e., as the anchoring point for the SC but possibly also as a suppressor/regulator of retrotransposition.

Repeating Marx

DEFF Research Database (Denmark)

Fuchs, Christian; Monticelli, Lara

2018-01-01

This introduction sets out the context of the special issue “Karl Marx @ 200: Debating Capitalism & Perspectives for the Future of Radical Theory”, which was published on the occasion of Marx’s bicentenary on 5 May 2018. First, we give a brief overview of contemporary capitalism’s development...... and its crises. Second, we argue that it is important to repeat Marx today. Third, we reflect on lessons learned from 200 years of struggles for alternatives to capitalism. Fourth, we give an overview of the contributions in this special issue. Taken together, the contributions in this special issue show...... that Marx’s theory and politics remain key inspirations for understanding exploitation and domination in 21st-century society and for struggles that aim to overcome these phenomena and establishing a just and fair society. We need to repeat Marx today....

Functional characterisation of the Schizosaccharomyces pombe homologue of the leukaemia-associated translocation breakpoint binding protein translin and its binding partner, TRAX.

Science.gov (United States)

Jaendling, Alessa; Ramayah, Soshila; Pryce, David W; McFarlane, Ramsay J

2008-02-01

Translin is a conserved protein which associates with the breakpoint junctions of chromosomal translocations linked with the development of some human cancers. It binds to both DNA and RNA and has been implicated in mRNA metabolism and regulation of genome stability. It has a binding partner, translin-associated protein X (TRAX), levels of which are regulated by the translin protein in higher eukaryotes. In this study we find that this regulatory function is conserved in the lower eukaryotes, suggesting that translin and TRAX have important functions which provide a selective advantage to both unicellular and multi-cellular eukaryotes, indicating that this function may not be tissue-specific in nature. However, to date, the biological importance of translin and TRAX remains unclear. Here we systematically investigate proposals that suggest translin and TRAX play roles in controlling mitotic cell proliferation, DNA damage responses, genome stability, meiotic/mitotic recombination and stability of GT-rich repeat sequences. We find no evidence for translin and/or TRAX primary function in these pathways, indicating that the conserved biochemical function of translin is not implicated in primary pathways for regulating genome stability and/or segregation.

Feature Binding of Common Everyday Items Is Not Affected by Age

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Serge Hoefeijzers

2017-05-01

Full Text Available There is a surge of studies confirming that old age spares the ability to bind in visual working memory (VWM multiple features within singular object representations. Furthermore, it has been suggested that such ability may also be independent of the cultural background of the assessed individual. However, this evidence has been gathered with tasks that use arbitrary bindings of unfamiliar features. Whether age spares memory binding functions when the memoranda are features of everyday life objects remains less well explored. The present study investigated the influence of age, memory delay, and education, on conjunctive binding functions responsible for representing everyday items in VWM. We asked 32 healthy young and 41 healthy older adults to perform a memory binding task. During the task, participants saw visual arrays of objects, colours, or coloured objects presented for 6 s. Immediately after they were asked either to select the objects or the colours that were presented during the study display from larger sets of objects or colours, or to recombine them by selecting from such sets the objects and their corresponding colours. This procedure was repeated immediately after but this time providing a 30 s unfiled delay. We manipulated familiarity by presenting congruent and incongruent object-colour pairings. The results showed that the ability to bind intrinsic features in VWM does not decline with age even when these features belong to everyday items and form novel or well-known associations. Such preserved memory binding abilities held across memory delays. The impact of feature congruency on item-recognition appears to be greater in older than in younger adults. This suggests that long-term memory (LTM supports binding functions carried out in VWM for familiar everyday items and older adults still benefit from this LTM support. We have expanded the evidence supporting the lack of age effects on VWM binding functions to new feature and

Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand.

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Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M; Iverson, T M

2016-10-11

Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life-threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays with SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To improve our understanding of the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpA BR ) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-Lewis X ), and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpA BR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpA BR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that binding of SrpA to platelets either is multivalent or occurs via a larger, disialylated glycan.

Altered SPECT 123I iomazenil Binding in the Cingulate Cortex of Children with Anorexia Nervosa

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Shinichiro eNagamitsu

2016-02-01

Full Text Available Several lines of evidence suggest that anxiety plays a key role in the development and maintenance of anorexia nervosa (AN in children. The purpose of this study was to examine cortical GABA(A-benzodiazepine receptor binding before and after treatment in children beginning intensive AN treatment. Brain single photon emission computed tomography (SPECT measurements using 123I iomazenil, which binds to GABA(A-benzodiazepine receptors, was performed in 26 participants with AN who were enrolled in a multimodal treatment program. Sixteen of the 26 participants underwent a repeat SPECT scan immediately before discharge at conclusion of the intensive treatment program. Eating behavior and mood disturbances were assessed using Eating Attitudes Test with 26 items (EAT-26 and the short form of the Profile of Mood States (POMS. Clinical outcome scores were evaluated after a 1-year period. We examined association between relative iomazenil binding activity in cortical regions of interest (ROIs and psychometric profiles, and determined which psychometric profiles show interaction effects with brain regions. Further, we determined if binding activity could predict clinical outcome and treatment changes. Higher EAT-26 scores were significantly associated with lower iomazenil binding activity in the anterior posterior cingulate cortex (ACC. Higher POMS subscale scores were significantly associated with lower iomazenil binding activity in the left frontal, parietal cortex, and posterior cingulate cortex (PCC. Depression-Dejection, and Confusion POMS subscale scores, and total POMS score, showed interaction effects with brain regions in iomazenil binding activity. Decreased binding in the ACC and left parietal cortex was associated with poor clinical outcomes. Relative binding increases throughout the PCC and occipital gyrus were observed after weight gain in children with AN. These findings suggest that cortical GABAergic receptor binding is altered in children

Analysis of repeated measures data

CERN Document Server

Islam, M Ataharul

2017-01-01

This book presents a broad range of statistical techniques to address emerging needs in the field of repeated measures. It also provides a comprehensive overview of extensions of generalized linear models for the bivariate exponential family of distributions, which represent a new development in analysing repeated measures data. The demand for statistical models for correlated outcomes has grown rapidly recently, mainly due to presence of two types of underlying associations: associations between outcomes, and associations between explanatory variables and outcomes. The book systematically addresses key problems arising in the modelling of repeated measures data, bearing in mind those factors that play a major role in estimating the underlying relationships between covariates and outcome variables for correlated outcome data. In addition, it presents new approaches to addressing current challenges in the field of repeated measures and models based on conditional and joint probabilities. Markov models of first...

LTRs of Endogenous Retroviruses as a Source of Tbx6 Binding Sites.

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Yasuhiko, Yukuto; Hirabayashi, Yoko; Ono, Ryuichi

2017-01-01

Retrotransposons are abundant in mammalian genomes and can modulate the gene expression of surrounding genes by disrupting endogenous binding sites for transcription factors (TFs) or providing novel TFs binding sites within retrotransposon sequences. Here, we show that a (C/T)CACACCT sequence motif in ORR1A, ORR1B, ORR1C, and ORR1D, Long Terminal Repeats (LTRs) of MaLR endogenous retrovirus (ERV), is the direct target of Tbx6, an evolutionary conserved family of T-box TFs. Moreover, by comparing gene expression between control mice (Tbx6 +/-) and Tbx6-deficient mice (Tbx6 -/-), we demonstrate that at least four genes, Twist2, Pitx2, Oscp1 , and Nfxl1 , are down-regulated with Tbx6 deficiency. These results suggest that ORR1A, ORR1B, ORR1C and ORR1D may contribute to the evolution of mammalian embryogenesis.

CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

DEFF Research Database (Denmark)

1995-01-01

Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

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

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

2011-01-01

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

Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

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Reinhardt, H Christian; Yaffe, Michael B

2013-09-01

Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

Saturable binding of 35S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

International Nuclear Information System (INIS)

Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

1984-01-01

35 -S-t-Butylbicyclophosphorothionate ( 35 S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25 0 C, in 250 mM NaCl, specific binding of 35 S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. 35 S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase 3 H-GABA binding. Pharmacological studies show that 35 S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of 35 S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of 35 S-TBPS in cortical membranes are compared. The present bindings are consistent with the interpretation that 35 S-TBPS binds, at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

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Emanuela Leonardi

Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

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Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

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David Piñeiro

2015-04-01

Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYB transcription factor in the regulation of trichome patterning in Arabidopsis

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Gan Lijun

2011-12-01

Full Text Available Abstract Background Single-repeat R3 MYB transcription factors (single-repeat MYBs play important roles in controlling trichome patterning in Arabidopsis. It was proposed that single-repeat MYBs negatively regulate trichome formation by competing with GLABRA1 (GL1 for binding GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3, thus inhibiting the formation of activator complex TTG1(TRANSPARENT TESTA GLABRA1-GL3/EGL3-GL1 that is required for the activation of GLABRA2 (GL2, whose product is a positive regulator of trichome formation. Previously we identified a novel single-repeat MYB transcription factor, TRICHOMELESS1 (TCL1, which negatively regulates trichome formation on the inflorescence stems and pedicels by directly suppressing the expression of GL1. Results We analyzed here the role of TRICHOMELESS2 (TCL2, a previously-uncharacterized single-repeat MYB transcription factor in trichome patterning in Arabidopsis. We showed that TCL2 is closely related to TCL1, and like TCL1 and other single-repeat MYBs, TCL2 interacts with GL3. Overexpression of TCL2 conferred glabrous phenotype while knockdown of TCL2 via RNAi induced ectopic trichome formation on the inflorescence stems and pedicels, a phenotype that was previously observed in tcl1 mutants. These results suggested that TCL2 may have overlapping function with TCL1 in controlling trichome formation on inflorescences. On the other hand, although the transcription of TCL2, like TCL1, is not controlled by the activator complex formed by GL1 and GL3, and TCL2 and TCL1 proteins are more than 80% identical at the amino acid level, the expression of TCL2 under the control of TCL1 promoter only partially recovered the mutant phenotype of tcl1, implying that TCL2 and TCL1 are not fully functional equivalent. Conclusions TCL2 function redundantly with TCL1 in controlling trichome formation on inflorescences, but they are not fully functional equivalent. Transcription of TCL2 is not controlled by activator complex

Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts

Science.gov (United States)

Jam, Faidruz Azura; Ismail, Zahariah; Wan Ngah, Wan Zurinah

2013-01-01

Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging. PMID:24396567

Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts

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Suzana Makpol

2013-01-01

Full Text Available Biodynes, tocotrienol-rich fraction (TRF, and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2 exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P<0.05. Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P<0.05 with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P<0.05. These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging.

Palm Tocotrienol-Rich Fraction Improves Vascular Proatherosclerotic Changes in Hyperhomocysteinemic Rats

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Ku-Zaifah Norsidah

2013-01-01

Full Text Available This study investigated the effects of palm tocotrienol-rich fraction (TRF on aortic proatherosclerotic changes in rats fed with a high methionine diet. Forty-two male Wistar rats were divided into six groups. The first group was the control (fed with a basal diet. Another five groups were fed with 1% methionine diet for 10 weeks. From week 6 onward, folate (8 mg/kg diet or palm TRF (30, 60, and 150 mg/kg diets was added into the diet of the last four rat groups, respectively. The high methionine diet raised the plasma total homocysteine and aortic lipid peroxidation, which were reduced by the palm TRF and folate supplementations. Plasma nitric oxide was reduced in the high methionine group compared to the control (3.72±0.57 versus 6.65±0.53 μmol/L, P<0.05, which reduction was reversed by the palm TRF (60 and 150 mg/kg and folate supplementations. The increased aortic vascular cell adhesion molecule-1 expression in the methionine group (2.58±0.29 was significantly reduced by the folate (1.38±0.18 and palm TRF at 150 mg/kg (1.19±0.23. Palm TRF was comparable to folate in reducing high methionine diet-induced plasma hyperhomocysteinemia, aortic oxidative stress, and inflammatory changes in rats.

Association of Circulating Transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle.

Science.gov (United States)

Casas, Eduardo; Cai, Guohong; Kuehn, Larry A; Register, Karen B; McDaneld, Tara G; Neill, John D

2018-03-13

High throughput sequencing allows identification of small non-coding RNAs. Transfer RNA Fragments are a class of small non-coding RNAs, and have been identified as being involved in inhibition of gene expression. Given their role, it is possible they may be involved in mediating the infection-induced defense response in the host. Therefore, the objective of this study was to identify 5' transfer RNA fragments (tRF5s) associated with a serum antibody response to M. bovis in beef cattle. The tRF5s encoding alanine, glutamic acid, glycine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with antibody response against M. bovis. tRF5s encoding alanine, glutamine, glutamic acid, glycine, histidine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with season, which could be attributed to calf growth. There were interactions (P < 0.05) between antibody response to M. bovis and season for tRF5 encoding selenocysteine (anticodon UGA), proline (anticodon CGG), and glutamine (anticodon TTG). Selenocysteine is a rarely used amino acid that is incorporated into proteins by the opal stop codon (UGA), and its function is not well understood. Differential expression of tRF5s was identified between ELISA-positive and negative animals. Production of tRF5s may be associated with a host defense mechanism triggered by bacterial infection, or it may provide some advantage to a pathogen during infection of a host. Further studies are needed to establish if tRF5s could be used as a diagnostic marker of chronic exposure.

Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells

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Raktim Biswas

2011-01-01

Full Text Available Crude ethanolic extract of Thuja occidentalis (Fam: Cupressaceae is used as homeopathic mother tincture (TOΦ to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C10H16O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell. Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

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Stefanie Hausammann

Full Text Available Inhibitory antibodies directed against coagulation factor VIII (FVIII can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Human telomeres are hypersensitive to UV-induced DNA Damage and refractory to repair.

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Patrick J Rochette

2010-04-01

Full Text Available Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV, the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD which are both mutagenic and lethal. The human telomeric repeat unit (5'TTAGGG/CCCTAA3' is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP-based technique, immunoprecipitation of DNA damage (IPoD, to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat.

Time-restricted feeding improves insulin resistance and hepatic steatosis in a mouse model of postmenopausal obesity.

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Chung, Heekyung; Chou, Winjet; Sears, Dorothy D; Patterson, Ruth E; Webster, Nicholas J G; Ellies, Lesley G

2016-12-01

Menopause is associated with significant hormonal changes that result in increased total body fat and abdominal fat, amplifying the risk for metabolic syndrome and diseases such as diabetes, cardiovascular disease and cancer in postmenopausal women. Intermittent fasting regimens hold significant health benefit promise for obese humans, however, regimens that include extreme daytime calorie restriction or daytime fasting are generally associated with hunger and irritability, hampering long-term compliance and adoption in the clinical setting. Time-restricted feeding (TRF), a regimen allowing eating only during a specific period in the normal circadian feeding cycle, without calorie restriction, may increase compliance and provide a more clinically viable method for reducing the detrimental metabolic consequences associated with obesity. We tested TRF as an intervention in a mouse model of postmenopausal obesity. Metabolic parameters were measured using Clinical Laboratory Animal Monitoring System (CLAMS) and we carried out glucose tolerance tests. We also stained liver sections with oil red O to examine steatosis and measured gene expression related to gluconeogenesis. Preexisting metabolic disease was significantly attenuated during 7 weeks of TRF. Despite having access to the same high fat diet (HFD) as ad libitum fed (ALF) mice, TRF mice experienced rapid weight loss followed by a delayed improvement in insulin resistance and a reduced severity of hepatic steatosis by having access to the HFD for only 8h during their normal nocturnal feeding period. The lower respiratory exchange ratio in the TRF group compared with the ALF group early in the dark phase suggested that fat was the predominant fuel source in the TRF group and correlated with gene expression analyses that suggested a switch from gluconeogenesis to ketogenesis. In addition, TRF mice were more physically active than ALF fed mice. Our data support further analysis of TRF as a clinically viable form of

Anticonvulsant effect of time-restricted feeding in a pilocarpine-induced seizure model: Metabolic and epigenetic implications.

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Jorge eLandgrave-Gómez

2016-01-01

Full Text Available A new generation of antiepileptic drugs has emerged; however, one-third of epilepsy patients do not properly respond to pharmacological treatments. The purpose of the present study was to investigate whether time-restricted feeding has an anticonvulsant effect and whether this restrictive diet promotes changes in energy metabolism and epigenetic modifications in a pilocarpine-induced seizure model. To resolve our hypothesis, one group of rats had free access to food and water ad libitum (AL and a second group underwent a time-restricted feeding (TRF schedule. We used the lithium-pilocarpine model to induce status epilepticus (SE, and behavioral seizure monitoring was analyzed. Additionally, an electroencephalography (EEG recording was performed to verify the effect of TRF on cortical electrical activity after a pilocarpine injection. For biochemical analysis, animals were sacrificed 24 hours after SE and hippocampal homogenates were used to evaluate the proteins related to metabolism and chromatin structure. Our results showed that TRF had an anticonvulsant effect as measured by the prolonged latency of forelimb clonus seizure, a decrease in the seizure severity score and fewer animals reaching SE. Additionally, the power of the late phase EEG recordings in the AL group was significantly higher than the TRF group. Moreover, we found that TRF is capable of inducing alterations in signaling pathways that regulate energy metabolism, including an increase in the phosphorylation of AMP dependent kinase (AMPK and a decrease in the phosphorylation of Akt kinase. Furthermore, we found that TRF was able to significantly increase the beta hydroxybutyrate (β-HB concentration, an endogenous inhibitor of histone deacetylases (HDACs. Finally, we found a significant decrease in HDAC activity as well as an increase in acetylation on histone 3 (H3 in hippocampal homogenates from the TRF group. These findings suggest that alterations in energy metabolism and the

simple sequence repeats (EST-SSR)

African Journals Online (AJOL)

Yomi

2012-01-19

Jan 19, 2012 ... 212 primer pairs selected, based on repeat patterns of n≥8 for di-, tri-, tetra- and penta-nucleotide repeat ... Cluster analysis revealed a high genetic similarity among the sugarcane (Saccharum spp.) breeding lines which could reduce the genetic gain in ..... The multiple allele characteristic of SSR com-.

Nifty Nines and Repeating Decimals

Science.gov (United States)

Brown, Scott A.

2016-01-01

The traditional technique for converting repeating decimals to common fractions can be found in nearly every algebra textbook that has been published, as well as in many precalculus texts. However, students generally encounter repeating decimal numerals earlier than high school when they study rational numbers in prealgebra classes. Therefore, how…

Repeated Prescribed Burning in Aspen

Science.gov (United States)

Donald A. Perala

1974-01-01

Infrequent burning weather, low flammability of the aspen-hardwood association, and prolific sprouting and seeding of shrubs and hardwoods made repeated dormant season burning a poor tool to convert good site aspen to conifers. Repeat fall burns for wildlife habitat maintenance is workable if species composition changes are not important.

Specific assay measuring binding of /sup 125/I-Gp 120 from HIV to T4/sup +//CD4/sup +/ cells

Energy Technology Data Exchange (ETDEWEB)

Lundin, K.; Nygren, A.; Ramstedt, U.; Gidlund, M.; Wigzell, H.; Arthur, L.O.; Robey, W.G.; Morein, B.

1987-02-26

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4/sup +/ (T4/sup +/) cells. /sup 125/I-Gp120 could be shown to selectively bind to CD4/sup +/ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4/sup +/ cells. Monoclonal antibodies to other cell surface components do not interfere with /sup 125/I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block /sup 125/I-GP120 binding, though with variable titers. The authors believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding. (Auth.). 24 refs.; 2 figs.; 8 tabs.

Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo.

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Christine R Langlois

2016-11-01

Full Text Available Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.

Characterization of α-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis

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Palittapongarnpim Prasit

2009-06-01

Full Text Available Abstract Background Alpha-isopropylmalate synthase (α-IPMS is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding α-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR. The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two α-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His6-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units. Results The recombinant His6-α-IPMS proteins with two and 14 copies (α-IPMS-2CR and α-IPMS-14CR, respectively of the repeat units were purified by immobilized metal ion affinity chromatography and gel filtration. Both enzymes were found to be dimers by gel filtration. Both enzymes work well at pH values of 7–8.5 and temperatures of 37–42°C. However, α-IPMS-14CR tolerates pH values and temperatures outside of this range better than α-IPMS-2CR does. α-IPMS-14CR has higher affinity than α-IPMS-2CR for the two substrates, α-ketoisovalerate and acetyl CoA. Furthermore, α-IPMS-2CR was feedback inhibited by the end product l-leucine, whereas α-IPMS-14CR was not. Conclusion The differences in the kinetic properties and the l-leucine feedback inhibition between the two M. tuberculosis α-IPMS proteins containing low and high numbers of VNTR indicate that a large VNTR insertion affects protein structure and function. Demonstration of l-leucine binding to α-IPMS-14CR would confirm whether or not α-IPMS-14CR responds to end-product feedback inhibition.

NC1 domain of type VII collagen binds to the beta3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.

Science.gov (United States)

Chen, M; Marinkovich, M P; Jones, J C; O'Toole, E A; Li, Y Y; Woodley, D T

1999-02-01

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al, 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the beta3 chain of laminin 5, but also to the gamma2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the beta3 chain. In contrast, the NC1-laminin 5 interaction was not affected by a monoclonal antibody to the alpha3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1-laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita.

Short consensus repeat domains extend the E-selectin structure in order to grab cells out of flow

KAUST Repository

Aleisa, Fajr A

2017-01-08

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. They are composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR (also called “sushi” domains), a transmembrane domain and a C-terminal cytoplasmic tail. The adhesion of cells (expressing ligands) to the endothelium (expressing the selection i.e., E-selectin) occurs through the interaction between the lectin domain of selectins and sLeX presenting ligands. Structural/function studies to date have mainly focused on investigating the influence of the lectin domain of E-selectin on its ability to bind its ligands while other domains received less atention. We prepared a number of different recombinant E-selectin proteins with changes in the SCR units. Specifically we generated wild-type E-selectin proteins as monomeric or dimeric structures, mutant proteins with varied numbers of SCRs as well as proteins where strategic residues were mutated to change the conformation of the selectin. Using a novel real time immunoprecipitation surface plasmon resonance (SPR)-based in vitro binding study developed in our lab, the interaction of recombinant E-selectin proteins with immunoprecipitated endogenous ligands (i.e. CD44) captured on a CM-5 chip was assessed. These studies provided quantitative binding kinetics with on and off rates of selectin-ligand interactions and suggested that robust binding is dependent on the presence of the SCRs and oligomerization. These results provide significant implications on the functional mechanism of E-selectin binding to its ligands.

Short consensus repeat domains extend the E-selectin structure in order to grab cells out of flow

KAUST Repository

Aleisa, Fajr A; Sakashita, Kosuke; Lee, Jaeman; Abu Samra, Dina Bashir Kamil; Habuchi, Satoshi; Kusakabe, Takahiro; Merzaban, Jasmeen

2017-01-01

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. They are composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR (also called “sushi” domains), a transmembrane domain and a C-terminal cytoplasmic tail. The adhesion of cells (expressing ligands) to the endothelium (expressing the selection i.e., E-selectin) occurs through the interaction between the lectin domain of selectins and sLeX presenting ligands. Structural/function studies to date have mainly focused on investigating the influence of the lectin domain of E-selectin on its ability to bind its ligands while other domains received less atention. We prepared a number of different recombinant E-selectin proteins with changes in the SCR units. Specifically we generated wild-type E-selectin proteins as monomeric or dimeric structures, mutant proteins with varied numbers of SCRs as well as proteins where strategic residues were mutated to change the conformation of the selectin. Using a novel real time immunoprecipitation surface plasmon resonance (SPR)-based in vitro binding study developed in our lab, the interaction of recombinant E-selectin proteins with immunoprecipitated endogenous ligands (i.e. CD44) captured on a CM-5 chip was assessed. These studies provided quantitative binding kinetics with on and off rates of selectin-ligand interactions and suggested that robust binding is dependent on the presence of the SCRs and oligomerization. These results provide significant implications on the functional mechanism of E-selectin binding to its ligands.

Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

Science.gov (United States)

Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

2014-09-15

Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Inhibitory effects of ginseng total saponin on up-regulation of cAMP pathway induced by repeated administration of morphine.

Science.gov (United States)

Seo, Jeong-Ju; Lee, Jae-Woong; Lee, Wan-Kyu; Hong, Jin-Tae; Lee, Chong-Kil; Lee, Myung-Koo; Oh, Ki-Wan

2008-02-01

We have reported that ginseng total saponin (GTS) inhibited the development of physical and psychological dependence on morphine. However, the possible molecular mechanisms of GTS are unclear. Therefore, this study was undertaken to understand the possible molecular mechanism of GTS on the inhibitory effects of morphine-induced dependence. It has been reported that the up-regulated cAMP pathway in the LC of the mouse brain after repeated administration of morphine contributes to the feature of withdrawals. GTS inhibited up-regulation of cAMP pathway in the LC after repeated administration of morphine in this experiment. GTS inhibited cAMP levels and protein expression of protein kinase A (PKA). In addition, GTS inhibited the increase of cAMP response element binding protein (CREB) phosphorylation. Therefore, we conclude that the inhibitory effects of GTS on morphine-induced dependence might be mediated by the inhibition of cAMP pathway.

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

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Janet M. Doolittle-Hall

2015-11-01

Full Text Available Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV, hepatitis B virus (HBV or Merkel cell polyomavirus (MCPyV. These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

The Pentapeptide Repeat Proteins

OpenAIRE

Vetting, Matthew W.; Hegde, Subray S.; Fajardo, J. Eduardo; Fiser, Andras; Roderick, Steven L.; Takiff, Howard E.; Blanchard, John S.

2006-01-01

The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F]-[S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Myc...

LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

Directory of Open Access Journals (Sweden)

Lisnyak Yu. V.

2017-10-01

algorithm. To model the reduction in flexibility of allosteric pocket on modulator binding, the unperturbed normal modes are first calculated for the protein. The calculation is then repeated, each time perturbing one of the pockets in the protein. These results are combined with output from Fpocket in a support vector machine (SVM to predict allosteric pockets on proteins. The AlloSite server is similar to the AlloPred method in that it uses the Fpocket algorithm to elucidate allosteric pockets, whereas AlloPred uses an approach that combines flexibility with the Fpocket output. Results and discussion. By computational solvent mapping method FTSite, we have explored M.tuberculosis urease nonamer surface to find sites that tend to bind small organic molecular probes representing fragments of drug molecules with diverse hydrophobic and hydrophilic properties. The predicted three top ranked binding sites were situated at the interfaces between chains C and A, and chain G of neighbour trimer (and at equivalent locations in symmetrical trimers as well. A mapping of enzymes generally yields the most probable sites situated in a subsite of the enzyme active site. This was not the case for MTU which active sites were inaccessible for probes due to the closed conformation of the covering flap, and predicted binding sites were located not far from them at the entrance into a deep pocket. To explore their possible structural and functional role, we correlated the locations of predicted MTU binding sites and its ancillary pockets (which remain open and solvent exposed even while the flap is closed and indicated their partial overlapping. This overlapping may suggest that predicted sites are likely the intermediate binding sites responsible for recruiting a ligand to another binding site deeply buried in the protein. To examine the possibility that predicted binding sites are the sites for allostery binding we carried out the search for probable sites of allostery binding on MTU

Regional GABA concentration and [3H]-diazepam binding in rat brain following repeated electroconvulsive shock

International Nuclear Information System (INIS)

Bowdler, J.M.; Green, A.R.; Minchin, M.C.W.; Nutt, D.J.

1983-01-01

It has been confirmed that 24 hours following a series of electroconvulsive shocks (ECS) given once daily for 10 days (ECS x 10) to rats there is an increase in GABA concentration in the corpus striatum. A similar change was seen after the ECS had been given to rats anaesthetised with halothane, or when 5 ECS were given spread out over 10 days, the rats being anaesthetised during the ECS. A daily convulsion for 10 days elicited by flurothyl exposure resulted in an increased striatal GABA concentration, but also increased the GABA concentration in the hypothalamus, hippocampus and cortex. The increase in striatal GABA concentration was present 24 hours after ECS daily for 5 days or 3 days after ECS daily for 10 days. No change in [ 3 H]-diazepam binding was seen in hippocampus, cortex or corpus striatum 24 hours after the last of 10 once daily ECS. The increase in striatal GABA concentration was therefore seen at all times when enhanced monoaminemediated behaviours have been demonstrated following seizures. (Author)

Conformational dynamics and ligand binding in the multi-domain protein PDC109.

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Hyun Jin Kim

2010-02-01

Full Text Available PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2 repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs, a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1, estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

Differences in gene expression of cells growing in conventional 2D versus 3D cell culture

International Nuclear Information System (INIS)

Zschenker, Oliver; Cordes, Nils; Streichert, Thomas

2009-01-01

Full text: Telomeres are DNA protein complexes on the ends of chromosomes that distinguish the ends of chromosomes from double strand breaks and prevent degradation or fusion by nonhomologous end-joining. The loss of telomeres is associated with a loss of heterochromatic features leading to a less compact chromatin structure which allows e.g. DNA repair proteins to get better access to the site of the DNA damage and facilitate chromosome fusions. Telomerase is an enzyme that can counteract the loss of telomeres by adding telomeric repeats on the ends of chromosomes. Since telomerase is active in most tumor cells, telomerase is suggested to be the reason for the unlimited number of cell divisions of cancer cells. TRF2 is one of the most important proteins of the Shelterin complex protecting the telomeres from shortening by inhibiting ATM which is up-stream of the DNA repair mechanisms. Thus, we are concentrating on TRF2 and telomerase to investigate the differences in DNA repair in telomeric (heterochromatic) versus euchromatic regions. Human cancer cells with differences in status of p53 and telomerase like A549, UT-SCC15 and FaDu cells are used. Without any treatment, FaDu cells express high levels of telomerase and TRF2 in conventional 2D cell culture which is in contrast to e.g. A549. We found that telomerase is even higher expressed in 3D than in 2D cell culture. To connect telomere associated processes to both repair of radiogenic DNA damage/lesions and to cell-extracellular matrix interactions, we performed whole genome microarray analysis. By comparing the differential expression of genes associated with these three cell functions, we intend to yield new molecular insight into radiotherapy relevant tumor characteristics, particularly radioresistance and DNA damage response network processing. (author)

Manganese Exposure from Drinking Water and Children’s Classroom Behavior in Bangladesh

Science.gov (United States)

Khan, Khalid; Factor-Litvak, Pam; Wasserman, Gail A.; Liu, Xinhua; Ahmed, Ershad; Parvez, Faruque; Slavkovich, Vesna; Levy, Diane; Mey, Jacob; van Geen, Alexander

2011-01-01

Background: Evidence of neurological, cognitive, and neuropsychological effects of manganese (Mn) exposure from drinking water (WMn) in children has generated widespread public health concern. At elevated exposures, Mn has been associated with increased levels of externalizing behaviors, including irritability, aggression, and impulsivity. Little is known about potential effects at lower exposures, especially in children. Moreover, little is known regarding potential interactions between exposure to Mn and other metals, especially arsenic (As). Objectives: We conducted a cross-sectional study of 201 children to investigate associations of Mn and As in tube well water with classroom behavior among elementary school children, 8–11 years of age, in Araihazar, Bangladesh. Methods: Data on exposures and behavioral outcomes were collected from the participants at the baseline of an ongoing longitudinal study of child intelligence. Study children were rated by their school teachers on externalizing and internalizing items of classroom behavior using the standardized Child Behavior Checklist-Teacher’s Report Form (CBCL-TRF). Results: Log-transformed WMn was positively and significantly associated with TRF internalizing [estimated β = 0.82; 95% confidence interval (CI), 0.08–1.56; p = 0.03], TRF externalizing (estimated β = 2.59; 95% CI, 0.81–4.37; p =0.004), and TRF total scores (estimated β = 3.35; 95% CI, 0.86–5.83; p = 0.008) in models that adjusted for log-transformed water arsenic (WAs) and sociodemographic covariates. We also observed a positive monotonic dose–response relationship between WMn and TRF externalizing and TRF total scores among the participants of the study. We did not find any significant associations between WAs and various scales of TRF scores. Conclusion: These observations reinforce the growing concern regarding the neurotoxicologic effects of WMn in children. PMID:21493178

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

Science.gov (United States)

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

2016-03-14

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

Evolutionary Implications of Metal Binding Features in Different Species’ Prion Protein: An Inorganic Point of View

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Diego La Mendola

2014-05-01

Full Text Available Prion disorders are a group of fatal neurodegenerative conditions of mammals. The key molecular event in the pathogenesis of such diseases is the conformational conversion of prion protein, PrPC, into a misfolded form rich in β-sheet structure, PrPSc, but the detailed mechanistic aspects of prion protein conversion remain enigmatic. There is uncertainty on the precise physiological function of PrPC in healthy individuals. Several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ mainly through a domain composed by four to five repeats of eight amino acids. In addition to mammals, PrP homologues have also been identified in birds, reptiles, amphibians and fish. The globular domain of protein is retained in the different species, suggesting that the protein carries out an essential common function. However, the comparison of amino acid sequences indicates that prion protein has evolved differently in each vertebrate class. The primary sequences are strongly conserved in each group, but these exhibit a low similarity with those of mammals. The N-terminal domain of different prions shows tandem amino acid repeats with an increasing amount of histidine residues going from amphibians to mammals. The difference in the sequence affects the number of copper binding sites, the affinity and the coordination environment of metal ions, suggesting that the involvement of prion in metal homeostasis may be a specific characteristic of mammalian prion protein. In this review, we describe the similarities and the differences in the metal binding of different species’ prion protein, as revealed by studies carried out on the entire protein and related peptide fragments.

Altered SPECT 123I-iomazenil Binding in the Cingulate Cortex of Children with Anorexia Nervosa

Science.gov (United States)

Nagamitsu, Shinichiro; Sakurai, Rieko; Matsuoka, Michiko; Chiba, Hiromi; Ozono, Shuichi; Tanigawa, Hitoshi; Yamashita, Yushiro; Kaida, Hayato; Ishibashi, Masatoshi; Kakuma, Tatsuki; Croarkin, Paul E.; Matsuishi, Toyojiro

2016-01-01

Several lines of evidence suggest that anxiety plays a key role in the development and maintenance of anorexia nervosa (AN) in children. The purpose of this study was to examine cortical GABA(A)-benzodiazepine receptor binding before and after treatment in children beginning intensive AN treatment. Brain single-photon emission computed tomography (SPECT) measurements using 123I-iomazenil, which binds to GABA(A)-benzodiazepine receptors, was performed in 26 participants with AN who were enrolled in a multimodal treatment program. Sixteen of the 26 participants underwent a repeat SPECT scan immediately before discharge at conclusion of the intensive treatment program. Eating behavior and mood disturbances were assessed using Eating Attitudes Test with 26 items (EAT-26) and the short form of the Profile of Mood States (POMS). Clinical outcome scores were evaluated after a 1-year period. We examined association between relative iomazenil-binding activity in cortical regions of interest and psychometric profiles and determined which psychometric profiles show interaction effects with brain regions. Further, we determined if binding activity could predict clinical outcome and treatment changes. Higher EAT-26 scores were significantly associated with lower iomazenil-binding activity in the anterior and posterior cingulate cortex. Higher POMS subscale scores were significantly associated with lower iomazenil-binding activity in the left frontal, parietal cortex, and posterior cingulate cortex (PCC). “Depression–Dejection” and “Confusion” POMS subscale scores, and total POMS score showed interaction effects with brain regions in iomazenil-binding activity. Decreased binding in the anterior cingulate cortex and left parietal cortex was associated with poor clinical outcomes. Relative binding increases throughout the PCC and occipital gyrus were observed after weight gain in children with AN. These findings suggest that cortical GABAergic receptor binding is altered

In situ detection of tandem DNA repeat length

Energy Technology Data Exchange (ETDEWEB)

Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

1996-11-01

A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

Automated genotyping of dinucleotide repeat markers

Energy Technology Data Exchange (ETDEWEB)

Perlin, M.W.; Hoffman, E.P. [Carnegie Mellon Univ., Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)

1994-09-01

The dinucleotide repeats (i.e., microsatellites) such as CA-repeats are a highly polymorphic, highly abundant class of PCR-amplifiable markers that have greatly streamlined genetic mapping experimentation. It is expected that over 30,000 such markers (including tri- and tetranucleotide repeats) will be characterized for routine use in the next few years. Since only size determination, and not sequencing, is required to determine alleles, in principle, dinucleotide repeat genotyping is easily performed on electrophoretic gels, and can be automated using DNA sequencers. Unfortunately, PCR stuttering with these markers generates not one band for each allele, but a pattern of bands. Since closely spaced alleles must be disambiguated by human scoring, this poses a key obstacle to full automation. We have developed methods that overcome this obstacle. Our model is that the observed data is generated by arithmetic superposition (i.e., convolution) of multiple allele patterns. By quantitatively measuring the size of each component band, and exploiting the unique stutter pattern associated with each marker, closely spaced alleles can be deconvolved; this unambiguously reconstructs the {open_quotes}true{close_quotes} allele bands, with stutter artifact removed. We used this approach in a system for automated diagnosis of (X-linked) Duchenne muscular dystrophy; four multiplexed CA-repeats within the dystrophin gene were assayed on a DNA sequencer. Our method accurately detected small variations in gel migration that shifted the allele size estimate. In 167 nonmutated alleles, 89% (149/167) showed no size variation, 9% (15/167) showed 1 bp variation, and 2% (3/167) showed 2 bp variation. We are currently developing a library of dinucleotide repeat patterns; together with our deconvolution methods, this library will enable fully automated genotyping of dinucleotide repeats from sizing data.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

Energy Technology Data Exchange (ETDEWEB)

Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

International Nuclear Information System (INIS)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

2014-01-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

[3]tetrahydrotrazodone binding. Association with serotonin binding sites

International Nuclear Information System (INIS)

Kendall, D.A.; Taylor, D.P.; Enna, S.J.

1983-01-01

High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone

International Nuclear Information System (INIS)

Koenig, R.J.; Brent, G.A.; Warne, R.L.; Larsen, P.R.; Moore, D.D.

1987-01-01

Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. The authors have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. They show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

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Greicy H Goto

2015-08-01

Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

Science.gov (United States)

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

2017-09-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

Science.gov (United States)

Townsend, Philip D; Dixon, Christopher H; Slootweg, Erik J; Sukarta, Octavina C A; Yang, Ally W H; Hughes, Timothy R; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Goverse, Aska; Cann, Martin J

2018-03-02

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and Nb Glk1, a Golden2-like transcription factor. Rx1 binds to Nb Glk1 in vitro and in planta. Nb Glk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of Nb Glk1 for DNA in vitro. Nb Glk1 activates cellular responses to potato virus X, whereas Rx1 associates with Nb Glk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Digital repeat analysis; setup and operation.

Science.gov (United States)

Nol, J; Isouard, G; Mirecki, J

2006-06-01

Since the emergence of digital imaging, there have been questions about the necessity of continuing reject analysis programs in imaging departments to evaluate performance and quality. As a marketing strategy, most suppliers of digital technology focus on the supremacy of the technology and its ability to reduce the number of repeats, resulting in less radiation doses given to patients and increased productivity in the department. On the other hand, quality assurance radiographers and radiologists believe that repeats are mainly related to positioning skills, and repeat analysis is the main tool to plan training needs to up-skill radiographers. A comparative study between conventional and digital imaging was undertaken to compare outcomes and evaluate the need for reject analysis. However, digital technology still being at its early development stages, setting a credible reject analysis program became the major task of the study. It took the department, with the help of the suppliers of the computed radiography reader and the picture archiving and communication system, over 2 years of software enhancement to build a reliable digital repeat analysis system. The results were supportive of both philosophies; the number of repeats as a result of exposure factors was reduced dramatically; however, the percentage of repeats as a result of positioning skills was slightly on the increase for the simple reason that some rejects in the conventional system qualifying for both exposure and positioning errors were classified as exposure error. The ability of digitally adjusting dark or light images reclassified some of those images as positioning errors.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

Science.gov (United States)

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

International Nuclear Information System (INIS)

Dahabieh, Matthew S.; Ooms, Marcel; Malcolm, Tom; Simon, Viviana; Sadowski, Ivan

2011-01-01

Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.

Reproducibility of repeated measures of deuterium substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) binding in the human brain

International Nuclear Information System (INIS)

Logan, Jean; Fowler, Joanna S.; Volkow, Nora D.; Wang, Gene-Jack; MacGregor, Robert R.; Shea, Colleen

2000-01-01

The purpose of this study was to assess the reproducibility of repeated positron emission tomography (PET) measures of brain monoamine oxidase B (MAO B) using deuterium-substituted [ 11 C]L-deprenyl ([ 11 C]L-deprenyl-D2) in normal subjects and to validate the method used for estimating the kinetic constants from the irreversible 3-compartment model applied to the tracer binding. Five normal healthy subjects (age range 23-73 years) each received two PET scans with [ 11 C]L-deprenyl-D2. The time interval between scans was 7-27 days. Time-activity data from eight regions of interest and an arterial plasma input function was used to calculate λk 3 , a model term proportional to MAO B, and K 1 , the plasma to brain transfer constant that is related to blood flow. Linear (LIN) and nonlinear least-squares (NLLSQ) estimation methods were used to calculate the optimum model constants. A comparison of time-activity curves for scan 1 and scan 2 showed that the percent of change for peak uptake varied from -18.5 to 15.0% and that increases and decreases in uptake on scan 2 were associated with increases and decreases in the value of the arterial input of the tracer. Calculation of λk 3 showed a difference between scan 1 and scan 2 in the global value ranging between -6.97 and 4.5% (average -2.1±4.7%). The average percent change for eight brain regions for the five subjects was -2.84±7.07%. Values of λk 3 for scan 1 and scan 2 were highly correlated (r 2 =0.98; p 1 showed a significant correlation between scan 1 and scan 2 (r 2 =0.61; p 11 C]L-deprenyl-D2 varied between scan 1 and scan 2, driven by the differences in arterial tracer input. Application of a 3-compartment model to regional time-activity data and arterial input function yielded λk 3 values for scan 1 and scan 2 with an average difference of -2.84 ± 7.07%. Linear regression applied to values of λk 3 from the LIN and NLLSQ methods validated the use of the linear method for calculating λk 3

Disruption and functional analysis of six ORFs on chromosome XV: YOL117w, YOL115w ( TRF4), YOL114c, YOL112w ( MSB4), YOL111c and YOL072w.

Science.gov (United States)

Iwanejko, L; Smith, K N; Loeillet, S; Nicolas, A; Fabre, F

1999-10-01

We have carried out the systematic disruption of six ORFs on chromosome XV, of Saccharomyces cerevisiae using the long flanking homology technique to replace each with the KanMX cassette; we have also constructed plasmids containing replacement cassettes and cognate clones for each ORF. Disruption of three of the ORFs-YOL117w, YOL114c, and YOL112w (also known as MSB4)-does not result in any noteworthy phenotype with respect to temperature or nutritional requirements, but yol112w mutants with an additional disruption of YNL293w, which encodes a protein similar to Yol112w, exhibit a slow growth phenotype. The protein specified by YOL114c shares similarity with the human DS-1 protein. Disruption of YOL115w confers slow growth, cold sensitivity and poor sporulation; this ORF has been described elsewhere as TRF4, which encodes a topoisomerase I-related protein. Cells with disruptions of YOL111c, whose product is weakly similar to the human ubiquitin-like protein GdX, are slightly impaired in mating. Mutants disrupted for YOL072w, the predicted product of which is unrelated to any protein of known function, grow slowly, are cold-sensitive and sporulate with reduced efficiency. Copyright 1999 John Wiley & Sons, Ltd.

Coexistence of 3G repeaters with LTE base stations.

Science.gov (United States)

Yeo, Woon-Young; Lee, Sang-Min; Hwang, Gyung-Ho; Kim, Jae-Hoon

2013-01-01

Repeaters have been an attractive solution for mobile operators to upgrade their wireless networks at low cost and to extend network coverage effectively. Since the first LTE commercial deployment in 2009, many mobile operators have launched LTE networks by upgrading their 3G and legacy networks. Because all 3G frequency bands are shared with the frequency bands for LTE deployment and 3G mobile operators have an enormous number of repeaters, reusing 3G repeaters in LTE networks is definitely a practical and cost-efficient solution. However, 3G repeaters usually do not support spatial multiplexing with multiple antennas, and thus it is difficult to reuse them directly in LTE networks. In order to support spatial multiplexing of LTE, the role of 3G repeaters should be replaced with small LTE base stations or MIMO-capable repeaters. In this paper, a repeater network is proposed to reuse 3G repeaters in LTE deployment while still supporting multilayer transmission of LTE. Interestingly, the proposed network has a higher cluster throughput than an LTE network with MIMO-capable repeaters.

Repeatability & Workability Evaluation of SIGMOD 2009

KAUST Repository

Manegold, Stefan

2010-12-15

SIGMOD 2008 was the first database conference that offered to test submitters\\' programs against their data to verify the repeatability of the experiments published [1]. Given the positive feedback concerning the SIGMOD 2008 repeatability initiative, SIGMOD 2009 modified and expanded the initiative with a workability assessment.

Repeatability & Workability Evaluation of SIGMOD 2009

KAUST Repository

Manegold, Stefan; Manolescu, Ioana; Afanasiev, Loredana; Feng, Jieling; Gou, G.; Hadjieleftheriou, Marios; Harizopoulos, Stavros; Kalnis, Panos; Karanasos, Konstantinos; Laurent, Dominique; Lupu, M.; Onose, N.; Ré , C.; Sans, Virginie; Senellart, Pierre; Wu, T.; Shasha, Dennis E.

2010-01-01

SIGMOD 2008 was the first database conference that offered to test submitters' programs against their data to verify the repeatability of the experiments published [1]. Given the positive feedback concerning the SIGMOD 2008 repeatability initiative, SIGMOD 2009 modified and expanded the initiative with a workability assessment.

Effects of Aging and Tocotrienol-Rich Fraction Supplementation on Brain Arginine Metabolism in Rats

Directory of Open Access Journals (Sweden)

Musalmah Mazlan

2017-01-01

Full Text Available Accumulating evidence suggests that altered arginine metabolism is involved in the aging and neurodegenerative processes. This study sought to determine the effects of age and vitamin E supplementation in the form of tocotrienol-rich fraction (TRF on brain arginine metabolism. Male Wistar rats at ages of 3 and 21 months were supplemented with TRF orally for 3 months prior to the dissection of tissue from five brain regions. The tissue concentrations of L-arginine and its nine downstream metabolites were quantified using high-performance liquid chromatography and liquid chromatography tandem mass spectrometry. We found age-related alterations in L-arginine metabolites in the chemical- and region-specific manners. Moreover, TRF supplementation reversed age-associated changes in arginine metabolites in the entorhinal cortex and cerebellum. Multiple regression analysis revealed a number of significant neurochemical-behavioral correlations, indicating the beneficial effects of TRF supplementation on memory and motor function.

FRB 121102: A Starquake-induced Repeater?

Science.gov (United States)

Wang, Weiyang; Luo, Rui; Yue, Han; Chen, Xuelei; Lee, Kejia; Xu, Renxin

2018-01-01

Since its initial discovery, the fast radio burst (FRB) FRB 121102 has been found to be repeating with millisecond-duration pulses. Very recently, 14 new bursts were detected by the Green Bank Telescope during its continuous monitoring observations. In this paper, we show that the burst energy distribution has a power-law form which is very similar to the Gutenberg–Richter law of earthquakes. In addition, the distribution of burst waiting time can be described as a Poissonian or Gaussian distribution, which is consistent with earthquakes, while the aftershock sequence exhibits some local correlations. These findings suggest that the repeating FRB pulses may originate from the starquakes of a pulsar. Noting that the soft gamma-ray repeaters (SGRs) also exhibit such distributions, the FRB could be powered by some starquake mechanisms associated with the SGRs, including the crustal activity of a magnetar or solidification-induced stress of a newborn strangeon star. These conjectures could be tested with more repeating samples.

Identification of histone H4-like TAF in Schizosaccharomyces pombe as a protein that interacts with WD repeat-containing TAF

OpenAIRE

Mitsuzawa, Hiroshi; Ishihama, Akira

2002-01-01

The general transcription factor TFIID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). We previously identified two distinct WD repeat-containing TAFs, spTAF72 and spTAF73, in the fission yeast Schizosaccharomyces pombe. Here we report the identification of another S.pombe TAF, spTAF50, which is the S.pombe homolog of histone H4-like TAFs such as human TAF80, Drosophila TAF60 and Saccharomyces cerevisiae TAF60. spTAF50 was identified in a two-hybrid scre...

Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

Directory of Open Access Journals (Sweden)

Mykol Larvie

2012-01-01

Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

Dynamics of the excited state intramolecular charge transfer

International Nuclear Information System (INIS)

Joo, T.; Kim, C.H.

2006-01-01

The 6-dodecanoyl-2-dimethylaminonaphtalene (laurdan), a derivative of 6-propanoyl- 2-dimethylaminonaphthalene (prodan), has been used as a fluorescent probe in cell imaging, especially in visualizing the lipid rafts by the generalized polarization (GP) images, where GP=(I 440 -I 490 )/(I 440 +I 490 ) with I being the fluorescence intensity. The fluorescence spectrum of laurdan is sensitive to its dipolar environment due to the intramolecular charge transfer (ICT) process in S 1 state, which results in a dual emission from the locally excited (LE) and the ICT states. The ICT process and the solvation of the ICT state are very sensitive to the dipolar nature of the environment. In this work, the ICT of laurdan in ethanol has been studied by femtosecond time resolved fluorescence (TRF), especially TRF spectra measurement without the conventional spectral reconstruction method. TRF probes the excited states exclusively, a unique advantage over the pump/probe transient absorption technique, although time resolution of the TRF is generally lower than transient absorption and the TRF spectra measurement was possible only though the spectral reconstruction. Over the years, critical advances in TRF technique have been made in our group to achieve <50 fs time resolution with direct full spectra measurement capability. Detailed ICT and the subsequent solvation processes can be visualized unambiguously from the TRF spectra. Fig. 1 shows the TRF spectra of laurdan in ethanol at several time delays. Surprisingly, two bands at 433 and 476 nm are clearly visible in the TRF spectra of laurdan even at T = 0 fs. As time increases, the band at 476 nm shifts to the red while its intensity increases. The band at 433 nm also shifts slightly to the red, but loses intensity as time increases. The intensity of the 476 nm band reaches maximum at around 5 ps, where it is roughly twice as intense as that at 0 fs, and stays constant until lifetime decay is noticeable. The spectra were fit by

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

Science.gov (United States)

Izsvák, Zsuzsanna; Khare, Dheeraj; Behlke, Joachim; Heinemann, Udo; Plasterk, Ronald H; Ivics, Zoltán

2002-09-13

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

International Nuclear Information System (INIS)

Kobayashi, Haruo; Suzuki, Tadahiko; Sakamoto, Maki; Hashimoto, Wataru; Kashiwada, Keiko; Sato, Itaru; Akahori, Fumiaki; Satoh, Tetsuo

2007-01-01

Activity of acetylcholinesterase (AChE) and specific binding of [ 3 H]quinuclidinyl benzilate (QNB), [ 3 H]pirenzepine (PZP) and [ 3 H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [ 3 H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [ 3 H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected

Novel anti-HIV peptides containing multiple copies of artificially designed heptad repeat motifs

International Nuclear Information System (INIS)

Shi Weiguo; Qi Zhi; Pan Chungen; Xue Na; Debnath, Asim K.; Qie Jiankun; Jiang Shibo; Liu Keliang

2008-01-01

The peptidic anti-HIV drug T20 (Fuzeon) and its analog C34 share a common heptad repeat (HR) sequence, but they have different functional domains, i.e., pocket- and lipid-binding domains (PBD and LBD, respectively). We hypothesize that novel anti-HIV peptides may be designed by using artificial sequences containing multiple copies of HR motifs plus zero, one or two functional domains. Surprisingly, we found that the peptides containing only the non-natural HR sequences could significantly inhibit HIV-1 infection, while addition of PBD and/or LBD to the peptides resulted in significant improvement of anti-HIV-1 activity. These results suggest that these artificial HR sequences, which may serve as structural domains, could be used as templates for the design of novel antiviral peptides against HIV and other viruses with class I fusion proteins

Fibrillin binds calcium and is coded by cDNAs that reveal a multidomain structure and alternatively spliced exons at the 5[prime] end

Energy Technology Data Exchange (ETDEWEB)

Corson, G.M.; Chalberg, S.C.; Charbonneau, N.L.; Sakai, L.Y. (Oregon Health Sciences Univ., Portland (United States)); Dietz, H.C. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States))

1993-08-01

Fibrillin is an important structural protein of the extracellular matrix. It is a large cysteine-rich glycoprotein with extensive intrachain disulfide bonds, likely contributed by multiple EGF-like repeats. The authors have previously published 6.9 kb of FBN1 cDNA sequence. FBN1 cDNA clones that extend the sequence 3089 bp in the 5[prime] direction are described in this report. The deduced primary structure suggests that fibrillin in composed of multiple domains. The most predominant features the presence of 43 calcium binding EGF-like repeats. They demonstrate here that fibrillin molecules bind calcium. In addition, three alternatively spliced exons at the 5[prime] end are described. Analysis of 5.8 kb of surrounding genomic sequence revealed a 1.8-kb CpG island spanning the alternatively spliced exons and the next downstream exon. Since FBN1 is the gene responsible for Marfan syndrome, the information presented here will be useful in identifying new mutations and in understanding the function of fibrillin in the pathogenesis of the disease. 42 refs., 7 figs.

Topographical distribution of decrements and recovery in muscarinic receptors from rat brains repeatedly exposed to sublethal doses of soman

International Nuclear Information System (INIS)

Churchill, L.; Pazdernik, T.L.; Jackson, J.L.; Nelson, S.R.; Samson, F.E.; McDonough, J.H. Jr.

1984-01-01

[3H]Quinuclidinyl benzilate binding to rat brain muscarinic receptors decreased after repeated exposure to soman, a potent organophosphorus cholinesterase inhibitor. The topographical distribution of this decrement was analyzed by quantitative receptor autoradiography. After 4 weeks of soman, three times a week, quinuclidinyl benzilate binding decreased to 67 to 80% of control in frontal and parietal cortex, caudate-putamen, lateral septum, hippocampal body, dentate gyrus, superior colliculus, nucleus of the fifth nerve, and central grey. Minor or no decreases were observed in thalamic or hypothalamic nuclei, reticular formation, pontine nuclei, inferior colliculus, nucleus of the seventh nerve, and cerebellum. Scatchard analyses of saturation curves using frontal cortex sections from soman-treated rats revealed a decrease in maximal quinuclidinyl benzilate binding from that in control rats and a return toward control levels by 24 days without any significant change in affinity. These brain areas showing significant decrements in muscarinic receptors recovered with a similar time course. An estimate of the time for 50% recovery for some of the brain areas was 14 days for superior colliculus, 16 days for cortex, and 19 days for hippocampal body. The application of quantitative receptor autoradiography to analyze receptor alterations has been valuable in localizing the telencephalon as a region more susceptible to change in receptor concentration

Topographical distribution of decrements and recovery in muscarinic receptors from rat brains repeatedly exposed to sublethal doses of soman

Energy Technology Data Exchange (ETDEWEB)

Churchill, L.; Pazdernik, T.L.; Jackson, J.L.; Nelson, S.R.; Samson, F.E.; McDonough, J.H. Jr.

1984-08-01

(3H)Quinuclidinyl benzilate binding to rat brain muscarinic receptors decreased after repeated exposure to soman, a potent organophosphorus cholinesterase inhibitor. The topographical distribution of this decrement was analyzed by quantitative receptor autoradiography. After 4 weeks of soman, three times a week, quinuclidinyl benzilate binding decreased to 67 to 80% of control in frontal and parietal cortex, caudate-putamen, lateral septum, hippocampal body, dentate gyrus, superior colliculus, nucleus of the fifth nerve, and central grey. Minor or no decreases were observed in thalamic or hypothalamic nuclei, reticular formation, pontine nuclei, inferior colliculus, nucleus of the seventh nerve, and cerebellum. Scatchard analyses of saturation curves using frontal cortex sections from soman-treated rats revealed a decrease in maximal quinuclidinyl benzilate binding from that in control rats and a return toward control levels by 24 days without any significant change in affinity. These brain areas showing significant decrements in muscarinic receptors recovered with a similar time course. An estimate of the time for 50% recovery for some of the brain areas was 14 days for superior colliculus, 16 days for cortex, and 19 days for hippocampal body. The application of quantitative receptor autoradiography to analyze receptor alterations has been valuable in localizing the telencephalon as a region more susceptible to change in receptor concentration.

The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.

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Alexandre Ottaviani

2009-02-01

Full Text Available Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD, which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer-promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.

Novel expressed sequence tag- simple sequence repeats (EST ...

African Journals Online (AJOL)

Using different bioinformatic criteria, the SUCEST database was used to mine for simple sequence repeat (SSR) markers. Among 42,189 clusters, 1,425 expressed sequence tag- simple sequence repeats (EST-SSRs) were identified in silico. Trinucleotide repeats were the most abundant SSRs detected. Of 212 primer pairs ...

Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

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Yu-Ru Zhang

Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

Role of memory errors in quantum repeaters

International Nuclear Information System (INIS)

Hartmann, L.; Kraus, B.; Briegel, H.-J.; Duer, W.

2007-01-01

We investigate the influence of memory errors in the quantum repeater scheme for long-range quantum communication. We show that the communication distance is limited in standard operation mode due to memory errors resulting from unavoidable waiting times for classical signals. We show how to overcome these limitations by (i) improving local memory and (ii) introducing two operational modes of the quantum repeater. In both operational modes, the repeater is run blindly, i.e., without waiting for classical signals to arrive. In the first scheme, entanglement purification protocols based on one-way classical communication are used allowing to communicate over arbitrary distances. However, the error thresholds for noise in local control operations are very stringent. The second scheme makes use of entanglement purification protocols with two-way classical communication and inherits the favorable error thresholds of the repeater run in standard mode. One can increase the possible communication distance by an order of magnitude with reasonable overhead in physical resources. We outline the architecture of a quantum repeater that can possibly ensure intercontinental quantum communication

MicroRNA Regulation of Telomerase Reverse Transcriptase (TERT: Micro Machines Pull Strings of Papier-Mâché Puppets

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Ammad Ahmad Farooqi

2018-04-01

Full Text Available Substantial fraction of high-quality information is continuously being added into the existing pool of knowledge related to the biology of telomeres. Based on the insights gleaned from decades of research, it is clear that chromosomal stability needs a highly controlled and dynamic balance of DNA gain and loss in each terminal tract of telomeric repeats. Telomeres are formed by tandem repeats of TTAGGG sequences, which are gradually lost with each round of division of the cells. Targeted inhibition of telomerase to effectively induce apoptosis in cancer cells has attracted tremendous attention and overwhelmingly increasingly list of telomerase inhibitors truthfully advocates pharmacological significance of telomerase. Telomerase reverse transcriptase (TERT is a multi-talented and catalytically active component of the telomerase-associated protein machinery. Different proteins of telomerase-associated machinery work in a synchronized and orchestrated manner to ensure proper maintenance of telomeric length of chromosomes. Rapidly emerging scientific findings about regulation of TERT by microRNAs has revolutionized our understanding related to the biology of telomeres and telomerase. In this review, we have comprehensively discussed how different miRNAs regulate TERT in different cancers. Use of miRNA-based therapeutics against TERT in different cancers needs detailed research in preclinical models for effective translation of laboratory findings to clinically effective therapeutics.

Ocular surface sensitivity repeatability with Cochet-Bonnet esthesiometer.

Science.gov (United States)

Chao, Cecilia; Stapleton, Fiona; Badarudin, Ezailina; Golebiowski, Blanka

2015-02-01

To determine the repeatability of ocular surface threshold measurements using the Cochet-Bonnet esthesiometer on the same day and 3 months apart. Two separate studies were conducted to determine the repeatability of ocular surface threshold measurements made on the same day (n = 20 subjects) and 3 months apart (n = 29 subjects). The Cochet-Bonnet esthesiometer was used to measure corneal and inferior conjunctival thresholds using the ascending method of limits. The pressure exerted by the Cochet-Bonnet esthesiometer was determined using an analytical balance, for both the 0.08- and 0.12-mm-diameter filaments. This calibration was then used to convert filament length measurements to pressure. Repeatability was determined using a Bland and Altman analysis. The pressure exerted at each filament length differed between the two filament diameters. The measured pressure also differed from values provided by the manufacturer. Repeatability of threshold measurements at the central cornea was shown to be good, with better repeatability for same-day measurements (coefficient of repeatability [CoR] = ±0.23 g/mm²) than for those 3 months apart (CoR = ±0.52 g/mm²). Threshold measurements at the inferior conjunctiva, in contrast, were poorly repeatable (CoR = ±12.78 g/mm²). Cochet-Bonnet esthesiometry is repeatable when performed on the central cornea on the same day and 3 months apart, but this instrument is not recommended for conjunctival threshold measurements.

Safety of Repeated Yttrium-90 Radioembolization

International Nuclear Information System (INIS)

Lam, Marnix G. E. H.; Louie, John D.; Iagaru, Andrei H.; Goris, Michael L.; Sze, Daniel Y.

2013-01-01

Purpose: Repeated radioembolization (RE) treatments carry theoretically higher risk of radiation-induced hepatic injury because of the liver’s cumulative memory of previous exposure. We performed a retrospective safety analysis on patients who underwent repeated RE. Methods: From 2004 to 2011, a total of 247 patients were treated by RE. Eight patients (5 men, 3 women, age range 51–71 years) underwent repeated treatment of a targeted territory, all with resin microspheres (SIR-Spheres; Sirtex, Lane Cove, Australia). Adverse events were graded during a standardized follow-up. In addition, the correlation between the occurrence of RE-induced liver disease (REILD) and multiple variables was investigated in univariate and multivariate analyses in all 247 patients who received RE. Results: Two patients died shortly after the second treatment (at 84 and 107 days) with signs and symptoms of REILD. Both patients underwent whole liver treatment twice (cumulative doses 3.08 and 2.66 GBq). The other 6 patients demonstrated only minor toxicities after receiving cumulative doses ranging from 2.41 to 3.88 GBq. All patients experienced objective tumor responses. In the whole population, multifactorial analysis identified three risk factors associated with REILD: repeated RE (p = 0.036), baseline serum total bilirubin (p = 0.048), and baseline serum aspartate aminotransferase (p = 0.043). Repeated RE proved to be the only independent risk factor for REILD in multivariate analysis (odds ratio 9.6; p = 0.002). Additionally, the administered activity per target volume (in GBq/L) was found to be an independent risk factor for REILD, but only in whole liver treatments (p = 0.033). Conclusion: The risk of REILD appears to be elevated for repeated RE. Objective tumor responses were observed, but establishment of safety limits will require improvement in dosimetric measurement and prediction

Safety of Repeated Yttrium-90 Radioembolization

Energy Technology Data Exchange (ETDEWEB)

Lam, Marnix G. E. H.; Louie, John D. [Stanford University School of Medicine, Division of Interventional Radiology (United States); Iagaru, Andrei H.; Goris, Michael L. [Stanford University School of Medicine, Division of Nuclear Medicine (United States); Sze, Daniel Y., E-mail: dansze@stanford.edu [Stanford University School of Medicine, Division of Interventional Radiology (United States)

2013-10-15

Purpose: Repeated radioembolization (RE) treatments carry theoretically higher risk of radiation-induced hepatic injury because of the liver's cumulative memory of previous exposure. We performed a retrospective safety analysis on patients who underwent repeated RE. Methods: From 2004 to 2011, a total of 247 patients were treated by RE. Eight patients (5 men, 3 women, age range 51-71 years) underwent repeated treatment of a targeted territory, all with resin microspheres (SIR-Spheres; Sirtex, Lane Cove, Australia). Adverse events were graded during a standardized follow-up. In addition, the correlation between the occurrence of RE-induced liver disease (REILD) and multiple variables was investigated in univariate and multivariate analyses in all 247 patients who received RE. Results: Two patients died shortly after the second treatment (at 84 and 107 days) with signs and symptoms of REILD. Both patients underwent whole liver treatment twice (cumulative doses 3.08 and 2.66 GBq). The other 6 patients demonstrated only minor toxicities after receiving cumulative doses ranging from 2.41 to 3.88 GBq. All patients experienced objective tumor responses. In the whole population, multifactorial analysis identified three risk factors associated with REILD: repeated RE (p = 0.036), baseline serum total bilirubin (p = 0.048), and baseline serum aspartate aminotransferase (p = 0.043). Repeated RE proved to be the only independent risk factor for REILD in multivariate analysis (odds ratio 9.6; p = 0.002). Additionally, the administered activity per target volume (in GBq/L) was found to be an independent risk factor for REILD, but only in whole liver treatments (p = 0.033). Conclusion: The risk of REILD appears to be elevated for repeated RE. Objective tumor responses were observed, but establishment of safety limits will require improvement in dosimetric measurement and prediction.

Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts

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Suzana Makpol

2011-01-01

Full Text Available This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF in preventing cellular senescence of human diploid fibroblasts (HDFs. Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

The neuroprotective effects of tocotrienol rich fraction and alpha tocopherol against glutamate injury in astrocytes

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Thilaga Rati Selvaraju

2014-11-01

Full Text Available Tocotrienol rich fraction (TRF is an extract of palm oil, which consists of 25% alpha tocopherol (α-TCP and 75% tocotrienols. TRF has been shown to possess potent antioxidant, anti-inflammatory, anticancer, neuroprotection, and cholesterol lowering activities. Glutamate is the main excitatory amino acid neurotransmitter in the central nervous system of mammalian, which can be excitotoxic, and it has been suggested to play a key role in neurodegenerative disorders like Parkinson’s and Alzheimer’s diseases. In this present study, the effects of vitamin E (TRF and α-TCP in protecting astrocytes against glutamate injury were elucidated. Astrocytes induced with 180 mM of glutamate lead to significant cell death. However, glutamate mediated cytotoxicity was diminished via pre and post supplementation of TRF and α-TCP. Hence, vitamin E acted as a potent antioxidant agent in recovering mitochondrial injury due to elevated oxidative stress, and enhanced better survivability upon glutamate toxicity.  

Dental Fear in Children with Repeated Tooth Injuries.

Science.gov (United States)

Negovetić Vranić, Dubravka; Ivančić Jokić, Nataša; Bakarčić, Danko; Carek, Andreja; Rotim, Željko; Verzak, Željko

2016-06-01

Tooth injuries are serious clinical conditions. Some children experience dental trauma only once, while others are more prone to repeated tooth injuries. Repeated dental trauma occurs in 19.4% to 30% of patients. Pain and dental trauma are the most common reasons for fear and anxiety. The main objective of this study was to investigate how dental trauma, as well as repeated dental trauma affects the occurrence and development of dental fear in children. The study was conducted on a random sample of 147 subjects (88 boys and 59 girls) aged 5-8 and 9-12 years. Subjects in both age groups were divided into subroups without dental trauma, with one dental trauma and with repeated dental trauma. The validated Children’s Fear Survey Schedule – Dental Subscale was used on fear assessment. Results showed that only 12.2% of children without trauma, 33.3% with one trauma and 51.7% with repeated trauma were not afraid of injection. Older children had a significantly lower fear of injections, touch of an unknown person, choking, going to the hospital and people in white uniforms. Dentist was not the cause of fear in 65.5% of patients with repeated trauma. With each repeated injury of teeth, the degree of their fear of dental treatment was lower.

Erroneous Memories Arising from Repeated Attempts to Remember

Science.gov (United States)

Henkel, Linda A.

2004-01-01

The impact of repeated and prolonged attempts at remembering on false memory rates was assessed in three experiments. Participants saw and imagined pictures and then made repeated recall attempts before taking a source memory test. Although the number of items recalled increased with repeated tests, the net gains were associated with more source…

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

Science.gov (United States)

Buczek, Pawel; Horvath, Martin P

2006-06-23

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

Science.gov (United States)

Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

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

International Nuclear Information System (INIS)

Öberg, Christine; Belikov, Sergey

2012-01-01

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

Identification, variation and transcription of pneumococcal repeat sequences

Science.gov (United States)

2011-01-01

Background Small interspersed repeats are commonly found in many bacterial chromosomes. Two families of repeats (BOX and RUP) have previously been identified in the genome of Streptococcus pneumoniae, a nasopharyngeal commensal and respiratory pathogen of humans. However, little is known about the role they play in pneumococcal genetics. Results Analysis of the genome of S. pneumoniae ATCC 700669 revealed the presence of a third repeat family, which we have named SPRITE. All three repeats are present at a reduced density in the genome of the closely related species S. mitis. However, they are almost entirely absent from all other streptococci, although a set of elements related to the pneumococcal BOX repeat was identified in the zoonotic pathogen S. suis. In conjunction with information regarding their distribution within the pneumococcal chromosome, this suggests that it is unlikely that these repeats are specialised sequences performing a particular role for the host, but rather that they constitute parasitic elements. However, comparing insertion sites between pneumococcal sequences indicates that they appear to transpose at a much lower rate than IS elements. Some large BOX elements in S. pneumoniae were found to encode open reading frames on both strands of the genome, whilst another was found to form a composite RNA structure with two T box riboswitches. In multiple cases, such BOX elements were demonstrated as being expressed using directional RNA-seq and RT-PCR. Conclusions BOX, RUP and SPRITE repeats appear to have proliferated extensively throughout the pneumococcal chromosome during the species' past, but novel insertions are currently occurring at a relatively slow rate. Through their extensive secondary structures, they seem likely to affect the expression of genes with which they are co-transcribed. Software for annotation of these repeats is freely available from ftp://ftp.sanger.ac.uk/pub/pathogens/strep_repeats/. PMID:21333003

Nonparametric additive regression for repeatedly measured data

KAUST Repository

Carroll, R. J.; Maity, A.; Mammen, E.; Yu, K.

2009-01-01

We develop an easily computed smooth backfitting algorithm for additive model fitting in repeated measures problems. Our methodology easily copes with various settings, such as when some covariates are the same over repeated response measurements

Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

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John M Louis

Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Quantum key distribution with two-segment quantum repeaters

Energy Technology Data Exchange (ETDEWEB)

Kampermann, Hermann; Abruzzo, Silvestre; Bruss, Dagmar [Theoretische Physik III, Heinrich-Heine-Universitaet Duesseldorf (Germany)

2014-07-01

Quantum repeaters represent one possible way to achieve long-distance quantum key distribution. One way of improving the repeater rate and decreasing the memory coherence time is the usage of multiplexing. Motivated by the experimental fact that long-range connections are practically demanding, we extend the analysis of the quantum repeater multiplexing protocol to the case of short-range connections. We derive formulas for the repeater rate and we show that short-range connections lead to most of the benefits of a full-range multiplexing protocol. A less demanding QKD-protocol without quantum memories was recently introduced by Lo et al. We generalize this measurement-device-independent quantum key Distribution protocol to the scenario where the repeater Station contains also heralded quantum memories. We assume either single-photon sources or weak coherent pulse sources plus decay states. We show that it is possible to significantly outperform the original proposal, even in presence of decoherence of the quantum memory. We give formulas in terms of device imperfections i.e., the quantum bit error rate and the repeater rate.

Giardia telomeric sequence d(TAGGG)4 forms two intramolecular G-quadruplexes in K+ solution: effect of loop length and sequence on the folding topology.

Science.gov (United States)

Hu, Lanying; Lim, Kah Wai; Bouaziz, Serge; Phan, Anh Tuân

2009-11-25

Recently, it has been shown that in K(+) solution the human telomeric sequence d[TAGGG(TTAGGG)(3)] forms a (3 + 1) intramolecular G-quadruplex, while the Bombyx mori telomeric sequence d[TAGG(TTAGG)(3)], which differs from the human counterpart only by one G deletion in each repeat, forms a chair-type intramolecular G-quadruplex, indicating an effect of G-tract length on the folding topology of G-quadruplexes. To explore the effect of loop length and sequence on the folding topology of G-quadruplexes, here we examine the structure of the four-repeat Giardia telomeric sequence d[TAGGG(TAGGG)(3)], which differs from the human counterpart only by one T deletion within the non-G linker in each repeat. We show by NMR that this sequence forms two different intramolecular G-quadruplexes in K(+) solution. The first one is a novel basket-type antiparallel-stranded G-quadruplex containing two G-tetrads, a G x (A-G) triad, and two A x T base pairs; the three loops are consecutively edgewise-diagonal-edgewise. The second one is a propeller-type parallel-stranded G-quadruplex involving three G-tetrads; the three loops are all double-chain-reversal. Recurrence of several structural elements in the observed structures suggests a "cut and paste" principle for the design and prediction of G-quadruplex topologies, for which different elements could be extracted from one G-quadruplex and inserted into another.

Preferential binding of p53 tumor suppressor to p21 promoter sites that contain inverted repeats capable of forming cruciform structure

Czech Academy of Sciences Publication Activity Database

Coufal, Jan; Jagelská, Eva; Liao, J.C.C.; Brázda, Václav

2013-01-01

Roč. 441, č. 1 (2013), s. 83-85 ISSN 0006-291X R&D Projects: GA ČR(CZ) GAP301/11/2076; GA ČR(CZ) GBP206/12/G151 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : DNA-BINDING * SUPERCOILED DNA * EXPRESSION Subject RIV: BO - Biophysics Impact factor: 2.281, year: 2013

Deployment Repeatability

Science.gov (United States)

2016-08-31

large cohort of trials to spot unusual cases. However, deployment repeatability is inherently a nonlinear phenomenon, which makes modeling difficult...and GEMS tip position were both tracked during ground testing by a laser target tracking system. Earlier SAILMAST testing in 2005 [8] used...recalls the strategy used by SRTM, where a constellation of lights was installed at the tip of the boom and a modified star tracker was used to track tip

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

KAUST Repository

Mahfouz, Magdy M.

2011-01-24

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

R-loops: targets for nuclease cleavage and repeat instability.

Science.gov (United States)

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Repeat profile analysis in an x-ray department

International Nuclear Information System (INIS)

Bassey, C.E.; Ojo, O.O.; Akpabio, I.

1991-01-01

The repeat profile of an x-ray department in a developing country was analysed monthly between July 1989 and June 1990. Results showed an average repeat rate of 3.7% for the period of study. The main causes of film repetition were: equipment fault, 33.9%; radiographer's fault, 27.4%; film fault, 19.3%; processing fault, 10.8% and patient's fault, 8.6%. The average repeat rate in the first 6 months of study reduced by 50% in the last 6 months. This was due to the effectiveness of implementation of corrective actions. The overall repeat rate was found to correlate well with both the equipment fault and radiographer's fault with correlation coefficients, r, of 0.94 and 0.91, respectively. It is expected that a further reduction in the repeat rate will be obtained after the introduction of quality assurance programmes. (author)

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

Science.gov (United States)

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

2016-01-01

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

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

An Investigation of Immunogenicity of Chitosan-Based Botulinum Neurotoxin E Binding Domain Recombinant Candidate Vaccine via Mucosal Route

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Mohammad Javad Bagheripour

2017-01-01

Full Text Available Background and Objectives: Botulism syndrome is caused by serotypes A-G of neurotoxins of Clostridium genus. Neurotoxin binding domain is an appropriate vaccine candidate due to its immunogenic activity. In this study, the immunogenicity of chitosan-based botulinum neurotoxin E binding domain recombinant candidate vaccine was investigated via mucosal route of administration. Methods: In this experimental study, chitosan nanoparticles containing rBoNT/E protein were synthesized by ionic gelation method and were administered orally and intranasally to mice. After each administration, IgG antibody titer was measured by ELISA method. Finally, all groups were challenged with active botulinum neurotoxin type E. Data were analyzed using Duncan and repeated ANOVA tests. The significance level was considered as p0.05, even intranasal route reduced the immunogenicity.

Effects of single and repeated administration of 1,2,3,4-tetrahydroisoquinoline analogs on the binding of [11C]raclopride to dopamine D2 receptors in the mouse brain

International Nuclear Information System (INIS)

Ishiwata, K.; Senda, M.; Saitoh, T.; Taguchi, K.; Toda, J.; Sano, T.; Koyanagi, Y.

2001-01-01

We investigated the effects of intraperitoneal injection of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogs and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the binding of [ 11 C]raclopride to striatal dopamine D 2 receptors in mice. The binding of [ 11 C]raclopride, but not of [ 11 C]N-methylspiperone or [ 11 C]nemonapride with higher affinity, to the receptors was significantly decreased immediately after TIQ injection. Neither a dopamine transporter blocker induced such effect nor TIQ affected the dopamine transporter-radioligand binding. Among the compounds investigated, including parkinsonism-inducing TIQ and (R/S)-1-benzyl-TIQ, parkinsonism-preventing (R)- and (S)-1-methyl-TIQ, and probable N-methylated metabolites of TIQ and 1-methyl-TIQ, TIQ and (S)-1-methyl-TIQ had the strongest effect on the binding of [ 11 C]raclopride, and N-methylated derivatives showed less of an effect than the respective parent compounds. The decrease in the binding of [ 11 C]raclopride continued for 7 hours and was followed by an increase until 10 days after the single and subchronic administration of TIQ. These findings suggest that TIQ analogs profoundly stimulated dopamine release which resulted in the competitive inhibition of the binding of [ 11 C]raclopride to dopamine D 2 receptors, but did not induce degeneration of the receptors. (author)

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans

Science.gov (United States)

Magennis, Eugene P.; Francini, Nora; Mastrotto, Francesca; Catania, Rosa; Redhead, Martin; Fernandez-Trillo, Francisco; Bradshaw, David; Churchley, David; Winzer, Klaus; Alexander, Cameron

2017-01-01

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans–E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and β-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure–property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S

CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems

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Kira eMakarova

2014-04-01

Full Text Available CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold. Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and effector domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

Science.gov (United States)

Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

2005-01-01

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

International Nuclear Information System (INIS)

Zhao Cunyou; Chen Yali; Park, Jiyoung; Kim, Jae Bum; Tang Hong

2004-01-01

Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication

Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1

DEFF Research Database (Denmark)

Pedersen, Margit; Kilstrup, Mogens; Hammer, Karin

2006-01-01

Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P-late transcriptional start...... site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may...... be important for transcriptional activation of P-late. Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription...

Impaired recovery of brain muscarinic receptor sites following an adaptive down-regulation induced by repeated administration of diisopropyl fluorophosphate in aged rats

International Nuclear Information System (INIS)

Pintor, A.; Fortuna, S.; De Angelis, S.; Michalek, H.

1990-01-01

Potential age-related differences in the recovery rate of brain cholinesterase activity (ChE) and muscarinic acetylcholine receptor binding sites (mAChRs) following reduction induced by repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. Male 3- and 24-month old rats were s.c. injected with DFP on alternate days for 2 weeks and killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the hippocampus and striatum, but not in the cerebral cortex, of control rats there as a significant age-related decline of ChE activity and maximal density of 3H-QNB binding sites (Bmax). The repeated administration of DFP during the first week caused a syndrome of cholinergic stimulation both in aged and young rats. The syndrome was more pronounced, in terms of intensity and duration in aged than in young animals resulting in 40 and 12% mortality, respectively; during the second week the syndrome attenuated in the two age-groups. The percentage inhibition of brain ChE at the end of DFP treatment did not differ between young and surviving aged rats. The down-regulation of mACRs was present in the three brain regions of both young and age rats (from 20 to 40%). Factorial analysis of variance showed significant differences for age, recovery rate, and significant interaction between age and recovery rate, both for ChE and mAChRs in young rats the three brain areas

Impaired recovery of brain muscarinic receptor sites following an adaptive down-regulation induced by repeated administration of diisopropyl fluorophosphate in aged rats

Energy Technology Data Exchange (ETDEWEB)

Pintor, A.; Fortuna, S.; De Angelis, S.; Michalek, H. (Istituto Superiore di Sanita, Rome (Italy))

1990-01-01

Potential age-related differences in the recovery rate of brain cholinesterase activity (ChE) and muscarinic acetylcholine receptor binding sites (mAChRs) following reduction induced by repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. Male 3- and 24-month old rats were s.c. injected with DFP on alternate days for 2 weeks and killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the hippocampus and striatum, but not in the cerebral cortex, of control rats there as a significant age-related decline of ChE activity and maximal density of 3H-QNB binding sites (Bmax). The repeated administration of DFP during the first week caused a syndrome of cholinergic stimulation both in aged and young rats. The syndrome was more pronounced, in terms of intensity and duration in aged than in young animals resulting in 40 and 12% mortality, respectively; during the second week the syndrome attenuated in the two age-groups. The percentage inhibition of brain ChE at the end of DFP treatment did not differ between young and surviving aged rats. The down-regulation of mACRs was present in the three brain regions of both young and age rats (from 20 to 40%). Factorial analysis of variance showed significant differences for age, recovery rate, and significant interaction between age and recovery rate, both for ChE and mAChRs in young rats the three brain areas.

Chronic renal failure (CRF in children in Jugoslavia

Directory of Open Access Journals (Sweden)

Peco-Antić Amira

2003-01-01

Full Text Available The aim of this study was to analyse the demographic variables of chronic non-terminal (CRF and terminal (TRF renal failure patients (pts younger than 19 years treated in Serbia in June 2001. The prevalence of CRF pts was registered as 4,7 per million total population (pmtp or 14,1 per million child population (pmcp while corresponding values for TRF pts were 4,5 pmtp or 13,5 pmcp. The incidence of TRF pts during the period Jan.2000-Jan.2002 was 4,35 pmcp. Boys dominated only among CRF pts (34:14; 60,4% beeing between the ages of 6 and 19 yrs while at the time of diagnosis of HBI, 33,3 % of boys were yanger than 2 yrs.The causes of CRF were: reflux nephropathy 58,3%, congenital kidney disease 16,7%, familial/hereditary 14,6% glomerulonephritis 6,2% and Willms tu 4,1%. Reflux nephropathy was also the most common underlying disease of TRF accounted for 36,9% of total cases while glomerulonephritis was responsible for 23,9 %. Reflux nephropathy was associated with neural tube defect in 53,3% and with congenital lower urinary tract obstruction in 66,7%. The most of CRF (81,25% and TRF pts (95,6% were from Serbia, the others were from Monte Negro and Republic Srpska. The most of CRF (65% and TRF (80% pts were treated in University Children’s Hospital in Belgrade. Of CRF pts 46% had serum sreatinine 100-200 μmol/l, in 11% of pts it was 400-600 μmol/l and 2% of pts were in pre-terminal CRF. One third of CRF pts had proteinuria 150-500 mg/l, and second third had proteinuria greater of 1000 mg/l. Anemia was present in 54% of CRf pts, and arterial hypertension in 56%. Hemodialysis was dominant treatment modality for TRF pts and only 23,9% had functioning transplant. Conclusion: This is the first national study of demographic characteristics of pediatric CRF in Serbia. Since its prevalence is considerably lower than that in Western and North European countries the true prevalence is some what higher. The increasing incidence of pediatric TRF from 2

De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies.

Science.gov (United States)

Mitsuhashi, Kana; Ito, Daisuke; Mashima, Kyoko; Oyama, Munenori; Takahashi, Shinichi; Suzuki, Norihiro

2017-12-04

Aberrant RNA-binding proteins form the core of the neurodegeneration cascade in spectrums of disease, such as amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Six ALS-related molecules, TDP-43, FUS, TAF15, EWSR1, heterogeneous nuclear (hn)RNPA1 and hnRNPA2 are RNA-binding proteins containing candidate mutations identified in ALS patients and those share several common features, including harboring an aggregation-prone prion-like domain (PrLD) containing a glycine/serine-tyrosine-glycine/serine (G/S-Y-G/S)-motif-enriched low-complexity sequence and rich in glutamine and/or asparagine. Additinally, these six molecules are components of RNA granules involved in RNA quality control and become mislocated from the nucleus to form cytoplasmic inclusion bodies (IBs) in the ALS/FTD-affected brain. To reveal the essential mechanisms involved in ALS/FTD-related cytotoxicity associated with RNA-binding proteins containing PrLDs, we designed artificial RNA-binding proteins harboring G/S-Y-G/S-motif repeats with and without enriched glutamine residues and nuclear-import/export-signal sequences and examined their cytotoxicity in vitro. These proteins recapitulated features of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and components of RNA granules, and cytotoxicity instigation. These findings indicated that these artificial RNA-binding proteins mimicked features of ALS-linked molecules and allowed the study of mechanisms associated with gain of toxic functions related to ALS/FTD pathogenesis.

De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

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Ricardo Vera Bravo

2011-12-01

Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

Treatment-related fluctuations in guillain barre syndrome and the conundrum of additional cycles of plasmapheresis

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Subasree Ramakrishnan

2017-01-01

Full Text Available Introduction: In Guillain Barre syndrome (GBS, worsening of weakness or disability after initial period of recovery or stabilization is described as treatment-related fluctuations (TRF. Aim: This study aims to describe the clinical characteristics and outcome of six patients with GBS and TRF. Patients and Methods: Six patients with GBS fulfilling NINCDS criteria, evaluated at a tertiary care university hospital during 2008–2017, were diagnosed to have TRF. They form the basis of this report. Results: All patients were men and their mean age was 40 years. At presentation, mean duration of illness was 15 days; the illness had plateaued in three and progressive in other three patients. Two of the four patients had variant GBS. Initially, five patients were treated with large volume plasmapheresis (LVPP and one patient with methyl prednisolone. At 17–28 days after disease onset, three patients developed new neurologic deficits (bilateral facial paresis in two; paralytic ileus in one. Other three patients with worsening of limb weakness (medical research council sum score of >5 and disability (Hughes disability grade by ≥1 fulfilled Kleyweg's criteria for TRF. All the six patients were treated with the completion of five cycles or additional cycles of LVPP. Conclusion: Awareness about TRF is essential for correct diagnosis and management of patients with GBS.

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

Science.gov (United States)

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

2016-01-13

Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

History, rare, and multiple events of mechanical unfolding of repeat proteins

Science.gov (United States)

Sumbul, Fidan; Marchesi, Arin; Rico, Felix

2018-03-01

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

Science.gov (United States)

Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

2009-10-12

Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

Development of analog watch with minute repeater

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Okigami, Tomio; Aoyama, Shigeru; Osa, Takashi; Igarashi, Kiyotaka; Ikegami, Tomomi

A complementary metal oxide semiconductor with large scale integration was developed for an electronic minute repeater. It is equipped with the synthetic struck sound circuit to generate natural struck sound necessary for the minute repeater. This circuit consists of an envelope curve drawing circuit, frequency mixer, polyphonic mixer, and booster circuit made by using analog circuit technology. This large scale integration is a single chip microcomputer with motor drivers and input ports in addition to the synthetic struck sound circuit, and it is possible to make an electronic system of minute repeater at a very low cost in comparison with the conventional type.

RTEL1 Inhibits Trinucleotide Repeat Expansions and Fragility

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Aisling Frizzell

2014-03-01

Full Text Available Human RTEL1 is an essential, multifunctional helicase that maintains telomeres, regulates homologous recombination, and helps prevent bone marrow failure. Here, we show that RTEL1 also blocks trinucleotide repeat expansions, the causal mutation for 17 neurological diseases. Increased expansion frequencies of (CTG⋅CAG repeats occurred in human cells following knockdown of RTEL1, but not the alternative helicase Fbh1, and purified RTEL1 efficiently unwound triplet repeat hairpins in vitro. The expansion-blocking activity of RTEL1 also required Rad18 and HLTF, homologs of yeast Rad18 and Rad5. These findings are reminiscent of budding yeast Srs2, which inhibits expansions, unwinds hairpins, and prevents triplet-repeat-induced chromosome fragility. Accordingly, we found expansions and fragility were suppressed in yeast srs2 mutants expressing RTEL1, but not Fbh1. We propose that RTEL1 serves as a human analog of Srs2 to inhibit (CTG⋅CAG repeat expansions and fragility, likely by unwinding problematic hairpins.

Isolation of human simple repeat loci by hybridization selection.

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Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

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Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

2013-01-01

Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

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Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2007-11-01

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

Data and material of the Safe-Range-Inventory: An assistance tool helping to improve the charging infrastructure for electric vehicles.

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Carbon, Claus-Christian; Gebauer, Fabian

2017-10-01

The Safe-Range-Inventory (SRI) was constructed in order to help public authorities to improve the charging infrastructures for electric vehicles [1; 10.1016/j.trf.2017.04.011]. Specifically, the impact of fast (vs slow) charging stations on people's range anxiety was examined. Ninety-seven electric vehicle users from Germany (81 male; M age =46.3 years, SD =12.1) were recruited to participate in the experimental design. Statistical analyses were conducted using ANOVA for repeated measures to test for interaction effects of available charging stations and remaining range with the dependent variable range anxiety . The full data set is publicly available via https://osf.io/bveyw/ (Carbon and Gebauer, 2017) [2].