Sample records for tat selectively regulates

  1. SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy.

    PubMed

    Xu, Muyu; Moresco, James J; Chang, Max; Mukim, Amey; Smith, Davey; Diedrich, Jolene K; Yates, John R; Jones, Katherine A

    2018-05-23

    HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.

  2. Novel mechanism and factor for regulation by HIV-1 Tat.

    PubMed Central

    Zhou, Q; Sharp, P A

    1995-01-01

    Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression. Images PMID:7835343

  3. Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB

    PubMed Central

    Fröbel, Julia; Rose, Patrick; Lausberg, Frank; Blümmel, Anne-Sophie; Freudl, Roland; Müller, Matthias

    2012-01-01

    The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport of folded proteins, which harbour signal sequences with a conserved twin-arginine motif. Many Tat translocases comprise the three membrane proteins TatA, TatB and TatC. TatC was previously shown to be involved in recognizing twin-arginine signal peptides. Here we show that beyond recognition, TatC mediates the transmembrane insertion of a twin-arginine signal sequence, thereby translocating the signal sequence cleavage site across the bilayer. In the absence of TatB, this can lead to the removal of the signal sequence even from a translocation-incompetent substrate. Hence interaction of twin-arginine signal peptides with TatB counteracts their premature cleavage uncoupled from translocation. This capacity of TatB is not shared by the homologous TatA protein. Collectively our results suggest that TatC is an insertase for twin-arginine signal peptides and that translocation-proficient signal sequence recognition requires the concerted action of TatC and TatB. PMID:23250441

  4. Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB.

    PubMed

    Fröbel, Julia; Rose, Patrick; Lausberg, Frank; Blümmel, Anne-Sophie; Freudl, Roland; Müller, Matthias

    2012-01-01

    The twin-arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates the transmembrane transport of folded proteins, which harbour signal sequences with a conserved twin-arginine motif. Many Tat translocases comprise the three membrane proteins TatA, TatB and TatC. TatC was previously shown to be involved in recognizing twin-arginine signal peptides. Here we show that beyond recognition, TatC mediates the transmembrane insertion of a twin-arginine signal sequence, thereby translocating the signal sequence cleavage site across the bilayer. In the absence of TatB, this can lead to the removal of the signal sequence even from a translocation-incompetent substrate. Hence interaction of twin-arginine signal peptides with TatB counteracts their premature cleavage uncoupled from translocation. This capacity of TatB is not shared by the homologous TatA protein. Collectively our results suggest that TatC is an insertase for twin-arginine signal peptides and that translocation-proficient signal sequence recognition requires the concerted action of TatC and TatB.

  5. Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

    PubMed Central

    Gonzalez-Hernandez, Marta J.; Cavalcoli, James D.; Sartor, Maureen A.; Contreras-Galindo, Rafael; Meng, Fan; Dai, Manhong; Dube, Derek; Saha, Anjan K.; Gitlin, Scott D.; Omenn, Gilbert S.; Kaplan, Mark H.

    2014-01-01

    ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis. PMID:24872592

  6. CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

    PubMed Central

    Zhong, Yu; Smart, Eric J.; Weksler, Babette; Couraud, Pierre-Olivier; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    The blood-brain barrier (BBB) is the critical structure for preventing HIV trafficking into the brain. Specific HIV proteins, such as Tat protein, can contribute to the dysfunction of tight junctions at the BBB and HIV entry into the brain. Tat is released by HIV-1 infected cells and can interact with a variety of cell surface receptors activating several signal transduction pathways, including those localized in caveolae. The present study focused on the mechanisms of Tat-induced caveolae-associated Ras signaling at the level of the BBB. Treatment with Tat activated the Ras pathway in human brain microvascular endothelial cells (HBMEC). However, caveolin-1 silencing markedly attenuated these effects. Because the integrity of the brain endothelium is regulated by intercellular tight junctions, these structural elements of the BBB were also evaluated in the present study. Exposure to Tat diminished the expression of several tight junction proteins, namely, occludin, zonula occludens (ZO)-1, and ZO-2 in the caveolar fraction of HBMEC. These effects were effectively protected by pharmacological inhibition of the Ras signaling and by silencing of caveolin-1. The present data indicate the importance of caveolae-associated signaling in the disruption of tight junctions upon Tat exposure. They also demonstrate that caveolin-1 may constitute an early and critical modulator that controls signaling pathways leading to the disruption of tight junction proteins. Thus, caveolin-1 may provide an effective target to protect against Tat-induced HBMEC dysfunction and the disruption of the BBB in HIV-1-infected patients. PMID:18667611

  7. EZH2 phosphorylation regulates Tat-induced HIV-1 transactivation via ROS/Akt signaling pathway.

    PubMed

    Zhang, Hong-Sheng; Liu, Yang; Wu, Tong-Chao; Du, Guang-Yuan; Zhang, Feng-Juan

    2015-12-21

    EZH2 plays a major role in HIV-1 latency, however, the molecular linkage between Tat-induced HIV-1 transactivation and EZH2 activity is not fully understood. It was shown Tat induced HIV-1 transactivation through inhibiting EZH2 activity. Tat decreased the levels of H3K27me3 and EZH2 occupy at the long terminal repeat (LTR) of HIV-1. We further showed for the first time that transfected with Tat construct resulted in an increase in phosphorylated EZH2 (p-EZH2), mediated by active Akt. ROS/Akt-dependent p-EZH2 was correlated with Tat-induced transactivation. Our study reveals that novel mechanisms allow Tat-induced HIV-1 transactivation by ROS/Akt-dependent downregulating the EZH2 epigenetic silencing machinery. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein.

    PubMed

    Rahimian, Pejman; He, Johnny J

    2016-12-01

    HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

  9. Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

    PubMed Central

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J.; Robinson, Colin

    2012-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70–90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6–8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed. PMID:22190680

  10. Ligand-gated purinergic receptors regulate HIV-1 Tat and morphine related neurotoxicity in primary mouse striatal neuron-glia co-cultures.

    PubMed

    Sorrell, Mary E; Hauser, Kurt F

    2014-03-01

    Emerging evidence suggests that opioid drugs, such as morphine and heroin, can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that opioid drugs acting via μ opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. Key aspects of microglial function are regulated by the P2X family of ATP activated ligand-gated ion channels. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, which prompted us to investigate whether the cation-permeable P2X receptors contribute to the neurotoxic effects of HIV and morphine. To address this question, neuron survival, as well as other endpoints including changes in dendritic length, extracellular ATP levels, and intracellular calcium levels, were assayed in primary neuron-glia co-cultures from mouse striatum. Treatment with TNP-ATP, a non-selective P2X antagonist, prevented the neurotoxic effects of exposure to morphine and/or HIV Tat, or ATP alone, suggesting P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. Although P2X7, and perhaps P2X1, receptor activation decreases neuron survival, neither P2X1, P2X3, nor P2X7 selective receptor antagonists prevented Tat and/or morphine-induced neurotoxicity. These and other experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and provide circumstantial evidence implicating P2X4 receptors in particular. Our findings reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompanies neuroAIDS and opiate abuse.

  11. Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

    PubMed Central

    Quizon, Pamela M.; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M.; Zhan, Chang-Guo; Zhu, Jun

    2016-01-01

    Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission. PMID:27966610

  12. Assembling the Tat protein translocase

    PubMed Central

    Alcock, Felicity; Stansfeld, Phillip J; Basit, Hajra; Habersetzer, Johann; Baker, Matthew AB; Palmer, Tracy; Wallace, Mark I; Berks, Ben C

    2016-01-01

    The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes. DOI: http://dx.doi.org/10.7554/eLife.20718.001 PMID:27914200

  13. Genetic Evidence for a Tight Cooperation of TatB and TatC during Productive Recognition of Twin-Arginine (Tat) Signal Peptides in Escherichia coli

    PubMed Central

    Lausberg, Frank; Fleckenstein, Stefan; Kreutzenbeck, Peter; Fröbel, Julia; Rose, Patrick; Müller, Matthias; Freudl, Roland

    2012-01-01

    The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D+2)-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D+2) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D+2)-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment. PMID:22761916

  14. Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli.

    PubMed

    Lausberg, Frank; Fleckenstein, Stefan; Kreutzenbeck, Peter; Fröbel, Julia; Rose, Patrick; Müller, Matthias; Freudl, Roland

    2012-01-01

    The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.

  15. The evolution of subtype B HIV-1 tat in the Netherlands during 1985-2012.

    PubMed

    van der Kuyl, Antoinette C; Vink, Monique; Zorgdrager, Fokla; Bakker, Margreet; Wymant, Chris; Hall, Matthew; Gall, Astrid; Blanquart, François; Berkhout, Ben; Fraser, Christophe; Cornelissen, Marion

    2018-05-02

    For the production of viral genomic RNA, HIV-1 is dependent on an early viral protein, Tat, which is required for high-level transcription. The quantity of viral RNA detectable in blood of HIV-1 infected individuals varies dramatically, and a factor involved could be the efficiency of Tat protein variants to stimulate RNA transcription. HIV-1 virulence, measured by set-point viral load, has been observed to increase over time in the Netherlands and elsewhere. Investigation of tat gene evolution in clinical isolates could discover a role of Tat in this changing virulence. A dataset of 291 Dutch HIV-1 subtype B tat genes, derived from full-length HIV-1 genome sequences from samples obtained between 1985-2012, was used to analyse the evolution of Tat. Twenty-two patient-derived tat genes, and the control Tat HXB2 were analysed for their capacity to stimulate expression of an LTR-luciferase reporter gene construct in diverse cell lines, as well as for their ability to complement a tat-defective HIV-1 LAI clone. Analysis of 291 historical tat sequences from the Netherlands showed ample amino acid (aa) variation between isolates, although no specific mutations were selected for over time. Of note, however, the encoded protein varied its length over the years through the loss or gain of stop codons in the second exon. In transmission clusters, a selection against the shorter Tat86 ORF was apparent in favour of the more common Tat101 version, likely due to negative selection against Tat86 itself, although random drift, transmission bottlenecks, or linkage to other variants could also explain the observation. There was no correlation between Tat length and set-point viral load; however, the number of non-intermediate variants in our study was small. In addition, variation in the length of Tat did not significantly change its capacity to stimulate transcription. From 1985 till 2012, variation in the length of the HIV-1 subtype B tat gene is increasingly found in the Dutch

  16. Nucleolar protein trafficking in response to HIV-1 Tat: rewiring the nucleolus.

    PubMed

    Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

    2012-01-01

    The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

  17. Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus

    PubMed Central

    Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W.

    2012-01-01

    The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these

  18. Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

    PubMed Central

    Sonnemann, Kevin J.; Heun-Johnson, Hanke; Turner, Amy J.; Baltgalvis, Kristen A.; Lowe, Dawn A.; Ervasti, James M.

    2009-01-01

    Background The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. Methods and Findings Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm2 versus 12.8±0.9 N/cm2; PBS versus TAT). Conclusions These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin. PMID:19478831

  19. Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.

    PubMed

    Kreutzenbeck, Peter; Kröger, Carsten; Lausberg, Frank; Blaudeck, Natascha; Sprenger, Georg A; Freudl, Roland

    2007-03-16

    The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

  20. Tat-APE1/ref-1 protein inhibits TNF-{alpha}-induced endothelial cell activation

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

    Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung

    2008-03-28

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-{alpha}-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-{alpha}-induced monocyte adhesion and vascular cell adhesion molecule-1 expressionmore » in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.« less

  1. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    PubMed

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

  2. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    PubMed

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  3. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

    PubMed Central

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-01-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

  4. The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

    PubMed

    Ulfig, Agnes; Freudl, Roland

    2018-05-11

    The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

  5. Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling

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

    Oh, Somi; You, Eunae; Ko, Panseon

    Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 ormore » the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of β-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression. - Highlights: • Ablation of αTAT1 inhibits HCT116 colon cancer cell invasion. • αTAT1/acetylated microtubules regulate expression of Wnt1/β-catenin target genes. • Acetylated microtubules regulate cellular localization of β-catenin. • Loss of αTAT1 prevents Wnt1 from inducing β-catenin-dependent and -independent pathways.« less

  6. HIV Tat/P-TEFb Interaction: A Potential Target for Novel Anti-HIV Therapies.

    PubMed

    Asamitsu, Kaori; Fujinaga, Koh; Okamoto, Takashi

    2018-04-17

    Transcription is a crucial step in the life cycle of the human immunodeficiency virus type 1 (HIV 1) and is primarily involved in the maintenance of viral latency. Both viral and cellular transcription factors, including transcriptional activators, suppressor proteins and epigenetic factors, are involved in HIV transcription from the proviral DNA integrated within the host cell genome. Among them, the virus-encoded transcriptional activator Tat is the master regulator of HIV transcription. Interestingly, unlike other known transcriptional activators, Tat primarily activates transcriptional elongation and initiation by interacting with the cellular positive transcriptional elongation factor b (P-TEFb). In this review, we describe the molecular mechanism underlying how Tat activates viral transcription through interaction with P-TEFb. We propose a novel therapeutic strategy against HIV replication through blocking Tat action.

  7. Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice.

    PubMed

    Schier, Christina J; Marks, William D; Paris, Jason J; Barbour, Aaron J; McLane, Virginia D; Maragos, William F; McQuiston, A Rory; Knapp, Pamela E; Hauser, Kurt F

    2017-06-07

    remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans -activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1. Copyright © 2017 the authors 0270-6474/17/375759-12$15.00/0.

  8. Expression of HIV-Tat protein is associated with learning and memory deficits in the mouse

    PubMed Central

    Carey, Amanda N.; Sypek, Elizabeth I.; Singh, Harminder D.; Kaufman, Marc J.; McLaughlin, Jay P.

    2012-01-01

    HIV-Tat protein has been implicated in the pathogenesis of HIV-1 neurological complications (i.e., neuroAIDS), but direct demonstrations of the effects of Tat on behavior are limited. GT-tg mice with a doxycycline (Dox)-inducible and brain-selective tat gene coding for Tat protein were used to test the hypothesis that the activity of Tat in brain is sufficient to impair learning and memory processes. Western blot analysis of GT-tg mouse brains demonstrated an increase in Tat antibody labeling that seemed to be dependent on the dose and duration of Dox pretreatment. Dox-treated GT-tg mice tested in the Barnes maze demonstrated longer latencies to find an escape hole and displayed deficits in probe trial performance, versus uninduced GT-tg littermates, suggesting Tat-induced impairments of spatial learning and memory. Reversal learning was also impaired in Tat-induced mice. Tat-induced mice additionally demonstrated long-lasting (up to one month) deficiencies in novel object recognition learning and memory performance. Furthermore, novel object recognition impairment was dependent on the dose and duration of Dox exposure, suggesting that Tat exposure progressively mediated deficits. These experiments provide evidence that Tat protein expression is sufficient to mediate cognitive abnormalities seen in HIV-infected individuals. Moreover, the genetically engineered GT-tg mouse may be useful for improving our understanding of the neurological underpinnings of neuroAIDS-related behaviors. PMID:22197678

  9. TAT [Training and Technology.

    ERIC Educational Resources Information Center

    Oak Ridge Associated Universities, TN. Manpower Development Div.

    The Oak Ridge Associated Universities (ORAU) of Tennessee and the Nuclear Division of the Union Carbide Corporation established an industrial training program called Training and Technology (TAT) which was conducted at the Oak Ridge Y-12 plant. TAT instructors were provided by the regular work force of Union Carbide while ORAU provided the…

  10. Phosphorylation of Tat-interactive protein 60 kDa by protein kinase C epsilon is important for its subcellular localisation.

    PubMed

    Sapountzi, Vasileia; Logan, Ian R; Nelson, Glyn; Cook, Susan; Robson, Craig N

    2008-01-01

    Tat-interactive protein 60 kDa is a nuclear acetyltransferase that both coactivates and corepresses transcription factors and has a definitive function in the DNA damage response. Here, we provide evidence that Tat-interactive protein 60 kDa is phosphorylated by protein kinase C epsilon. In vitro, protein kinase C epsilon phosphorylates Tat-interactive protein 60 kDa on at least two sites within the acetyltransferase domain. In whole cells, activation of protein kinase C increases the levels of phosphorylated Tat-interactive protein 60 kDa and the interaction of Tat-interactive protein 60 kDa with protein kinase C epsilon. A phosphomimetic mutant Tat-interactive protein 60 kDa has distinct subcellular localisation compared to the wild-type protein in whole cells. Taken together, these findings suggest that the protein kinase C epsilon phosphorylation sites on Tat-interactive protein 60 kDa are important for its subcellular localisation. Regulation of the subcellular localisation of Tat-interactive protein 60 kDa via phosphorylation provides a novel means of controlling Tat-interactive protein 60 kDa function.

  11. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

    PubMed

    Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

    2014-01-01

    BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

  12. HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction

    PubMed Central

    Rom, Slava; Pacifici, Marco; Passiatore, Giovanni; Aprea, Susanna; Waligorska, Agnieszka; Valle, Luis Del; Peruzzi, Francesca

    2011-01-01

    The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia. PMID:21745501

  13. Didehydro-Cortistatin A inhibits HIV-1 Tat mediated neuroinflammation and prevents potentiation of cocaine reward in Tat transgenic mice

    PubMed Central

    Mediouni, Sonia; Jablonski, Joseph; Paris, Jason J.; Clementz, Mark A.; Thenin-Houssier, Suzie; McLaughlin, Jay P.; Valente, Susana T.

    2015-01-01

    HIV-1 Tat protein has been shown to have a crucial role in HIV-1-associated neurocognitive disorders (HAND), which includes a group of syndromes ranging from undetectable neurocognitive impairment to dementia. The abuse of psychostimulants, such as cocaine, by HIV infected individuals, may accelerate and intensify neurological damage. On the other hand, exposure to Tat potentiates cocaine-mediated reward mechanisms, which further promotes HAND. Here, we show that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, crosses the blood-brain barrier, cross-neutralizes Tat activity from several HIV-1 clades and decreases Tat uptake by glial cell lines. In addition, dCA potently inhibits Tat mediated dysregulation of IL-1β, TNF-α and MCP-1, key neuroinflammatory signaling proteins. Importantly, using a mouse model where doxycycline induces Tat expression, we demonstrate that dCA reverses the potentiation of cocaine-mediated reward. Our results suggest that adding a Tat inhibitor, such as dCA, to current antiretroviral therapy may reduce HIV-1-related neuropathogenesis. PMID:25613133

  14. Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury.

    PubMed

    Jeong, Hoon Jae; Kim, Dae Won; Kim, Mi Jin; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Hwang, Hyun Sook; Kim, Duk Soo; Cho, Sung Woo; Won, Moo Ho; Han, Kyu Hyung; Park, Jin Seu; Eum, Won Sik; Choi, Soo Young

    2012-10-31

    Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

  15. Relaxed evolution in the tyrosine aminotransferase gene tat in old world fruit bats (Chiroptera: Pteropodidae).

    PubMed

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

  16. HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas.

    PubMed

    Luzzi, Anna; Morettini, Federica; Gazaneo, Sara; Mundo, Lucia; Onnis, Anna; Mannucci, Susanna; Rogena, Emily A; Bellan, Cristiana; Leoncini, Lorenzo; De Falco, Giulia

    2014-01-01

    A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.

  17. Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

    PubMed

    Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

    2015-05-01

    Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

  18. Relaxed Evolution in the Tyrosine Aminotransferase Gene Tat in Old World Fruit Bats (Chiroptera: Pteropodidae)

    PubMed Central

    Shen, Bin; Fang, Tao; Yang, Tianxiao; Jones, Gareth; Irwin, David M.; Zhang, Shuyi

    2014-01-01

    Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet. PMID:24824435

  19. Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice.

    PubMed

    Fitting, Sylvia; Stevens, David L; Khan, Fayez A; Scoggins, Krista L; Enga, Rachel M; Beardsley, Patrick M; Knapp, Pamela E; Dewey, William L; Hauser, Kurt F

    2016-01-01

    Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence. Copyright © 2015 by The American Society for Pharmacology and Experimental

  20. Role of Tat-interacting protein of 110 kDa and microRNAs in the regulation of hematopoiesis.

    PubMed

    Liu, Ying; He, Johnny J

    2016-07-01

    Hematopoiesis is regulated by cellular factors including transcription factors, microRNAs, and epigenetic modifiers. Understanding how these factors regulate hematopoiesis is pivotal for manipulating them to achieve their desired potential. In this review, we will focus on HIV-1 Tat-interacting protein of 110 kDa (Tip110) and its regulation of hematopoiesis. There are several pathways in hematopoiesis that involve Tip110 regulation. Tip110 is expressed in human cord blood CD34 cells; its expression decreases when CD34 cells begin to differentiate. Tip110 is also expressed in mouse marrow hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC). Tip110 expression increases the number, survival, and cell cycling of HPC. Tip110-mediated regulation of hematopoiesis has been linked to its reciprocal control of proto-oncogene expression. Small noncoding microRNAs (miRs) have been shown to play important roles in regulation of hematopoiesis. miR-124 specifically targets 3'-untranslated region of Tip110 and subsequently regulates Tip110 expression in HSC. Our recent findings for manipulating expression levels of Tip110 in HSC and HPC could be useful for expanding HSC and HPC and for improving engraftment of cord blood HSC/HPC.

  1. The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.

    PubMed

    Ulfig, Agnes; Fröbel, Julia; Lausberg, Frank; Blümmel, Anne-Sophie; Heide, Anna Katharina; Müller, Matthias; Freudl, Roland

    2017-06-30

    The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N -oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Extensive interactions between HIV TAT and TAF(II)250.

    PubMed

    Weissman, J D; Hwang, J R; Singer, D S

    2001-03-09

    The HIV transactivator, Tat, has been shown to be capable of potent repression of transcription initiation. Repression is mediated by the C-terminal segment of Tat, which binds the TFIID component, TAF(II)250, although the site(s) of interaction were not defined previously. We now report that the interaction between Tat and TAF(II)250 is extensive and involves multiple contacts between the Tat protein and TAF(II)250. The C-terminal domain of Tat, which is necessary for repression of transcription initiation, binds to a segment of TAF(II)250 that encompasses its acetyl transferase (AT) domain (885-1034 amino acids (aa)). Surprisingly, the N-terminal segment of Tat, which contains its activation domains, also binds to TAF(II)250 and interacts with two discontinuous segments of TAF(II)250 located between 885 and 984 aa and 1120 and 1279 aa. Binding of Tat to the 885-984 aa segment of TAF(II)250 requires the cysteine-rich domain of Tat, but not the acidic or glutamine-rich domains. Binding by the N-terminal domain of Tat to the 1120-1279 aa TAF(II)250 segment does not involve the acidic, cysteine- or glutamine-rich domains. Repression of transcription initiation by Tat requires functional TAF(II)250. We now demonstrate that transcription of the HIV LTR does not depend on TAF(II)250 which may account for its resistance to Tat mediated repression.

  3. Sources of male chauvinism in the TAT.

    PubMed

    Potkay, C R; Merrens, M R

    1975-10-01

    Potential sources of antifemale bias in TAT stimuli were evaluated by having 358 undergraduate subjects rate 17 male and 17 female TAT figures on 7-point anchored scales. Data from the five independent rating conditions were examined by 2 x 2 ANOVA. Biases toward greater Mental Health and Intelligence for female figures were seen to be insufficient counterbalancers of biases toward greater Cultural Favorability and Identification for male figures. Achievement status was rated equivalently. TAT stimuli appeared to show a "built in" source of male chauvinism systematically "pulling" male-sex identification. Potential for unfavorable clinical evaluation was seen to be greater for female TAT subjects compared with male subjects.

  4. PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

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

    Zhu Xuhui; Department of Laboratory Medicine, Tongji Hospital and Tongji Medical College of Huazhong University of Science and Technology, Wuhan; Yao Honghong

    2009-10-15

    The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, therebymore » underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.« less

  5. Facilitated extinction of morphine conditioned place preference with Tat-GluA2(3Y) interference peptide.

    PubMed

    Dias, C; Wang, Y T; Phillips, A G

    2012-08-01

    Neuroplasticity including long-term depression (LTD) has been implicated in both learning processes and addiction. LTD can be blocked by intravenous administration of the interference peptide Tat-GluA2(3Y) that prevents regulated endocytosis of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor. In this study, Tat-GluA2(3Y) was used to assess the role of LTD in the induction, expression, extinction and reinstatement of morphine-induced conditioned place preference (CPP). CPP was established in rats by pairing morphine (5 mg/kg, i.p.) or saline with a specific environmental context using a balanced protocol. Tat-GluA2(3Y) (0; 1.5; 2.25 nmol/g; i.v.), scrambled peptide (Tat-GluA2(Sc)), or vehicle was administered during the acquisition phase or prior to the test for CPP. Tat-GluA2(3Y) had no effect on the induction or initial expression of morphine-induced CPP. Rats that received Tat-GluA2(3Y) or Tat-GluA2(Sc) during acquisition were subsequently tested for 11 consecutive days in order to extinguish morphine CPP. CPP was then reinstated by an injection of morphine (5 mg/kg, i.p.). Co-administration of morphine and Tat-GluA2(3Y) during acquisition greatly facilitated extinction of CPP without affecting morphine-induced reinstatement of CPP. Using an intermittent retest schedule with bi-weekly tests to measure the maintenance of CPP, Tat-GluA2(3Y) during the acquisition phase had no effect on the maintenance of CPP. We propose that co-administration of Tat-GluA2(3Y) with morphine during acquisition of CPP weakens the association between morphine and contextual cues leading to rapid extinction of morphine CPP with repeated daily testing. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

    PubMed Central

    Taylor, J P; Pomerantz, R; Bagasra, O; Chowdhury, M; Rappaport, J; Khalili, K; Amini, S

    1992-01-01

    The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1505523

  7. Human immunodeficiency virus-1 protein Tat induces excitotoxic loss of presynaptic terminals in hippocampal cultures.

    PubMed

    Shin, Angela H; Thayer, Stanley A

    2013-05-01

    Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin-proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. PATHOPHYSIOLOGICAL CONSEQUENCES OF TAT-HKII PEPTIDE ADMINISTRATION ARE INDEPENDENT OF IMPAIRED VASCULAR FUNCTION AND ENSUING ISCHEMIA

    PubMed Central

    Nederlof, Rianne; Xie, Chaoqin; Eerbeek, Otto; Koeman, Anneke; Milstein, Dan MJ; Hollmann, Markus W; Mik, Egbert G; Warley, Alice; Southworth, Richard; Akar, Fadi G.; Zuurbier, Coert J

    2013-01-01

    Rationale We have shown that partial dissociation of HKII from mitochondria in the intact heart using low dose (200 nM) TAT-HKII prevents the cardioprotective effects of ischemic preconditioning (IPC) whereas high-dose (10 μM) TAT-HKII administration results in rapid myocardial dysfunction, mitochondrial depolarization and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely due to vasoconstriction and ensuing ischemia. Objective To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. Methods and Results Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential (AP) mapping, analysis of lactate production, NADH epifluorescence, lactate dehydrogenase (LDH) release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of IPC, is not associated with ischemia or ischemic-injury. Conclusions Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia; thereby, lending further credence to the role of mitochondria bound HKII as a critical regulator of cardiac function, ischemia-reperfusion (IR) injury, and cardioprotection by IPC. PMID:23329797

  9. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

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

    Dabney-Smith, Carole

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembledmore » proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cpTat

  10. Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression.

    PubMed

    Huan, Chen; Li, Zhaolong; Ning, Shanshan; Wang, Hong; Yu, Xiao-Fang; Zhang, Wenyan

    2018-05-01

    The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4 + T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions. IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1. Copyright © 2018 American Society for Microbiology.

  11. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

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

    Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process bymore » recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.« less

  12. Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

    PubMed Central

    Kehn-Hall, Kylene; Guendel, Irene; Carpio, Lawrence; Skaltsounis, Leandros; Meijer, Laurent; Al-Harthi, Lena; Steiner, Joseph P.; Nath, Avindra; Kutsch, Olaf; Kashanchi, Fatah

    2013-01-01

    The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40 nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4 nM in primary macrophages and 0.5 nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03 nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity. PMID:21514616

  13. MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

    PubMed

    Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

    2017-01-01

    In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

  14. Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T-Cell Hyperactivation

    PubMed Central

    Kwon, Hye-Sook; Brent, Michael M.; Getachew, Ruth; Jayakumar, Prerana; Chen, Lin-Feng; Schnolzer, Martina; McBurney, Michael W.; Marmorstein, Ronen; Greene, Warner C.; Ott, Melanie

    2009-01-01

    Summary Symptoms of T-cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-κB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-κB-responsive genes, a function lost in SIRT1−/− cells. These results support a model where the normal function of SIRT1 as a negative regulator of T-cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals. PMID:18329615

  15. Anti-inflammatory effects of Tat-Annexin protein on ovalbumin-induced airway inflammation in a mouse model of asthma

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

    Lee, Sun Hwa; Kim, Dae Won; Kim, Hye Ri

    Highlights: Black-Right-Pointing-Pointer We construct a cell permeable Tat-ANX1 fusion protein. Black-Right-Pointing-Pointer We examined the protective effects of Tat-ANX1 protein on OVA-induced asthma in animal models. Black-Right-Pointing-Pointer Transduced Tat-ANX1 protein protects from the OVA-induced production of cytokines and eosinophils in BAL fluid. Black-Right-Pointing-Pointer Tat-ANX1 protein markedly reduced OVA-induced MAPK in lung tissues. Black-Right-Pointing-Pointer Tat-ANX1 protein could be useful as a therapeutic agent for lung disorders including asthma. -- Abstract: Chronic airway inflammation is a key feature of bronchial asthma. Annexin-1 (ANX1) is an anti-inflammatory protein that is an important modulator and plays a key role in inflammation. Although the precise actionmore » of ANX1 remains unclear, it has emerged as a potential drug target for inflammatory diseases such as asthma. To examine the protective effects of ANX1 protein on ovalbumin (OVA)-induced asthma in animal models, we used a cell-permeable Tat-ANX1 protein. Mice sensitized and challenged with OVA antigen had an increased amount of cytokines and eosinophils in their bronchoalveolar lavage (BAL) fluid. However, administration of Tat-ANX1 protein before OVA challenge significantly decreased the levels of cytokines (interleukin (IL)-4, IL-5, and IL-13) and BAL fluid in lung tissues. Furthermore, OVA significantly increased the activation of mitogen-activated protein kinase (MAPK) in lung tissues, whereas Tat-ANX1 protein markedly reduced phosphorylation of MAPKs such as extracellular signal-regulated protein kinase, p38, and stress-activated protein kinase/c-Jun N-terminal kinase. These results suggest that transduced Tat-ANX1 protein may be a potential protein therapeutic agent for the treatment of lung disorders including asthma.« less

  16. TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

    NASA Astrophysics Data System (ADS)

    Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

    2012-06-01

    Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

  17. HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors.

    PubMed

    Sforza, Fabio; Nicoli, Francesco; Gallerani, Eleonora; Finessi, Valentina; Reali, Eva; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

    2014-07-31

    HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

  18. Effects of the TAT peptide orientation and relative location on the protein transduction efficiency.

    PubMed

    Guo, Qingguo; Zhao, Guojie; Hao, Fengjin; Guan, Yifu

    2012-05-01

    To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT-EGFP conjugates: TAT(+)-EGFP, TAT(-)-EGFP, EGFP-TAT(+) and EGFP-TAT(-), where TAT(+) and TAT(-) represent the original and the reversed TAT sequence, respectively. These four TAT-EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT-EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT-EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications. © 2011 John Wiley & Sons A/S.

  19. FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

    PubMed

    Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

    2002-03-01

    FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

  20. FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles

    PubMed Central

    Pendergrast, P. Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

    2002-01-01

    FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription. PMID:11907272

  1. Oxidative Stress Is Associated with Neuroinflammation in Animal Models of HIV-1 Tat Neurotoxicity

    PubMed Central

    Louboutin, Jean-Pierre; Agrawal, Lokesh; Reyes, Beverly A. S.; Van Bockstaele, Elisabeth J.; Strayer, David S.

    2014-01-01

    HIV-1 trans-acting protein Tat, an essential protein for viral replication, is a key mediator of neurotoxicity. If Tat oxidant injury and neurotoxicity have been described, consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with Tat, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. Tat injection caused oxidative stress. Administration of Tat in the CP induced an increase in numbers of Iba-1- and CD68-positive cells, as well as an infiltration of astrocytes. We also tested the effect of more protracted Tat exposure on neuroinflammation using an experimental model of chronic Tat exposure. SV(Tat): a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of Tat, oxidative stress, and ongoing apoptosis, mainly located in neurons. Intra-CP SV(Tat) injection induced an increase in microglia and astrocytes, suggesting that protracted Tat production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced neuroinflammation following Tat administration into the CP. Thus, Tat-induced oxidative stress, CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery. PMID:26784879

  2. Development of Tat-Conjugated Dendrimer for Transdermal DNA Vaccine Delivery.

    PubMed

    Bahadoran, Azadeh; Moeini, Hassan; Bejo, Mohd Hair; Hussein, Mohd Zobir; Omar, Abdul Rahman

    In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed. First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry. TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-).  In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine.  The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05). The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  3. Biological activity of Tat (47-58) peptide on human pathogenic fungi.

    PubMed

    Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun

    2006-06-23

    Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

  4. Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

    PubMed Central

    Kim, Dong-Hyun; Kim, Baek

    2011-01-01

    The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

  5. Functional Tat transport of unstructured, small, hydrophilic proteins.

    PubMed

    Richter, Silke; Lindenstrauss, Ute; Lücke, Christian; Bayliss, Richard; Brüser, Thomas

    2007-11-16

    The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.

  6. Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

    PubMed

    Kim, Jiyoung; Kim, Yeon-Soo

    2018-06-06

    Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

  7. HIV-1 Tat-based vaccines: from basic science to clinical trials.

    PubMed

    Fanales-Belasio, Emanuele; Cafaro, Aurelio; Cara, Andrea; Negri, Donatella R M; Fiorelli, Valeria; Butto, Stefano; Moretti, Sonia; Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Tripiciano, Antonella; Sernicola, Leonardo; Scoglio, Arianna; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; Ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Vardas, Eftyhia; Magnani, Mauro; Laguardia, Elena; Caputo, Antonella; Titti, Fausto; Ensoli, Barbara

    2002-09-01

    Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.

  8. Biological activity of Tat (47-58) peptide on human pathogenic fungi

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

    Jung, Hyun Jun; Park, Yoonkyung; Department of Biotechnology, Chosun University, 375 Seosuk-dong, Kwangju 501-750

    2006-06-23

    Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol;more » 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.« less

  9. Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury

    PubMed Central

    Stevens, Patrick R.; Gawryluk, Jeremy W.; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D.

    2015-01-01

    HIV-1 infected individuals are living longer but experiencing a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells leads to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat1-72-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND. PMID:25613139

  10. Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury.

    PubMed

    Stevens, Patrick R; Gawryluk, Jeremy W; Hui, Liang; Chen, Xuesong; Geiger, Jonathan D

    2014-01-01

    HIV-1 infected individuals live longer but experience a prevalence rate of over 50% for HIV-1 associated neurocognitive disorders (HAND) for which no effective treatment is available. Viral and cellular factors secreted by HIV-1 infected cells lead to neuronal injury and HIV-1 Tat continues to be implicated in the pathogenesis of HAND. Here we tested the hypothesis that creatine protected against HIV-1 Tat-induced neuronal injury by preventing mitochondrial bioenergetic crisis and/or redox catastrophe. Creatine blocked HIV-1 Tat(1-72)-induced increases in neuron cell death and synaptic area loss. Creatine protected against HIV-1 Tat-induced decreases in ATP. Creatine and creatine plus HIV-1 Tat increased cellular levels of creatine, and creatine plus HIV-1 Tat further decreased ratios of phosphocreatine to creatine observed with creatine or HIV-1 Tat treatments alone. Additionally, creatine protected against HIV-1 Tat-induced mitochondrial hypopolarization and HIV-1 Tat-induced mitochondrial permeability transition pore opening. Thus, creatine may be a useful adjunctive therapy against HAND.

  11. Impaired plant growth and development caused by human immunodeficiency virus type 1 Tat.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.

  12. Gold nanoparticles mediated colorimetric assay for HIV-Tat protein detection

    NASA Astrophysics Data System (ADS)

    Hashwan, Saeed S. Ba; Ruslinda, A. Rahim; Fatin, M. F.; Gopinath, Subash C. B.; Thivina, V.; Tony, V. C. S.; Arshad, M. K. Md.; Hashim, U.

    2016-07-01

    Gold-nanoparticle (AuNP) based colorimetric assays have been formulated for different biomolecular interactions. With this assay the probe such as antibody immobilized on the Au surface and in the presence of appropriate binding partner (antigen), will interact with each other on the Au surface. By following this strategy, herein we formulated a detection system with two anti-HIV-Tat antibodies, Mono (McAb) - and polyclonal (PcAb) by immobilizing them independently with different AuNPs. Under this condition, these two antibodies are under dispersed condition, and in the presence of HIV-Tat antigen, these molecules will be connected and forms the aggregation of AuNPs. This strategy yield rapid results, can be monitored by the spectral changes in UV-Vis spectrophotometry. Experiments were performed with two different methods using two anti-HIV-Tats monoclonal and one Polyclonal antibody against the antigen HIV-Tat. Between these methods conjugation of HIV-Tat and McAb on the AuNP followed by addition of PcAb yielded better results.

  13. A strategy of win-stay, lose-shift that outperforms tit-for-tat in the Prisoner's Dilemma game

    NASA Astrophysics Data System (ADS)

    Nowak, Martin; Sigmund, Karl

    1993-07-01

    THE Prisoner's Dilemma is the leading metaphor for the evolution of cooperative behaviour in populations of selfish agents, especially since the well-known computer tournaments of Axelrod1 and their application to biological communities2,3. In Axelrod's simulations, the simple strategy tit-for-tat did outstandingly well and subsequently became the major paradigm for reciprocal altruism4 12. Here we present extended evolutionary simulations of heterogeneous ensembles of probabilistic strategies including mutation and selection, and report the unexpected success of another protagonist: Pavlov. This strategy is as simple as tit-for-tat and embodies the fundamental behavioural mechanism win-stay, lose-shift, which seems to be a widespread rule13. Pavlov's success is based on two important advantages over tit-for-tat: it can correct occasional mistakes and exploit unconditional cooperators. This second feature prevents Pavlov populations from being undermined by unconditional cooperators, which in turn invite defectors. Pavlov seems to be more robust than tit-for-tat, suggesting that cooperative behaviour in natural situations may often be based on win-stay, lose-shift.

  14. Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

    PubMed Central

    Sakane, Naoki; Kwon, Hye-Sook; Pagans, Sara; Kaehlcke, Katrin; Mizusawa, Yasuhiro; Kamada, Masafumi; Lassen, Kara G.; Chan, Jonathan; Greene, Warner C.; Schnoelzer, Martina; Ott, Melanie

    2011-01-01

    The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation

  15. In Vivo Selection of CD4+ T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

    PubMed Central

    Braun, Stephen E.; Taube, Ran; Zhu, Quan; Wong, Fay Eng; Murakami, Akikazu; Kamau, Erick; Dwyer, Markryan; Qiu, Gang; Daigle, Janet; Carville, Angela; Johnson, R. Paul

    2012-01-01

    Abstract We evaluated the potential of an anti–human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4+ cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4+ T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4+ cell. Upon reinfusion of engineered autologous CD4+ cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4+ T cells progressively decreased, concurrent with loss of CD4+ cells and elevated viral loads in both animals. However, CD4+ T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4+ cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo. PMID:22734618

  16. HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection.

    PubMed

    Fanales-Belasio, Emanuele; Moretti, Sonia; Fiorelli, Valeria; Tripiciano, Antonella; Pavone Cossut, Maria R; Scoglio, Arianna; Collacchi, Barbara; Nappi, Filomena; Macchia, Iole; Bellino, Stefania; Francavilla, Vittorio; Caputo, Antonella; Barillari, Giovanni; Magnani, Mauro; Laguardia, Maria Elena; Cafaro, Aurelio; Titti, Fausto; Monini, Paolo; Ensoli, Fabrizio; Ensoli, Barbara

    2009-03-01

    Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.

  17. Fruit-specific expression of the human immunodeficiency virus type 1 tat gene in tomato plants and its immunogenic potential in mice.

    PubMed

    Ramírez, Yuri Jorge Peña; Tasciotti, Ennio; Gutierrez-Ortega, Abel; Donayre Torres, Alberto J; Olivera Flores, María Teresa; Giacca, Mauro; Gómez Lim, Miguel Angel

    2007-06-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 microg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.

  18. The preventive phase I trial with the HIV-1 Tat-based vaccine.

    PubMed

    Ensoli, Barbara; Fiorelli, Valeria; Ensoli, Fabrizio; Lazzarin, Adriano; Visintini, Raffaele; Narciso, Pasquale; Di Carlo, Aldo; Tripiciano, Antonella; Longo, Olimpia; Bellino, Stefania; Francavilla, Vittorio; Paniccia, Giovanni; Arancio, Angela; Scoglio, Arianna; Collacchi, Barbara; Ruiz Alvarez, Maria Josè; Tambussi, Giuseppe; Tassan Din, Chiara; Palamara, Guido; Latini, Alessandra; Antinori, Andrea; D'Offizi, Gianpiero; Giuliani, Massimo; Giulianelli, Marina; Carta, Maria; Monini, Paolo; Magnani, Mauro; Garaci, Enrico

    2009-12-11

    The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

  19. Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

    PubMed

    Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

    2017-07-04

    Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

  20. Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

    PubMed Central

    Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A.S.; Zhou, Pengbo; Mesuraca, Maria

    2017-01-01

    Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes. PMID:28187462

  1. Safety and immunogenicity of HIV-1 Tat toxoid in immunocompromised HIV-1-infected patients.

    PubMed

    Gringeri, A; Santagostino, E; Muça-Perja, M; Mannucci, P M; Zagury, J F; Bizzini, B; Lachgar, A; Carcagno, M; Rappaport, J; Criscuolo, M; Blattner, W; Burny, A; Gallo, R C; Zagury, D

    1998-01-01

    To antagonize the deleterious effects of the HIV-1 toxin extracellular Tat on uninfected immune cells, we developed a new strategy of anti-HIV-1 vaccine using an inactivated but immunogenic Tat (Tat toxoid). Tat toxoid has been assayed for safety and immunogenicity in seropositive patients. The phase I vaccine clinical trial testing Tat toxoid preparation in Seppic Isa 51 oil adjuvant was performed on 14 HIV-1-infected asymptomatic although biologically immunocompromised individuals (500-200 CD4+ cells/mm3). Following as many as 8 injections, no clinical defects were observed. All patients exhibited an antibody (Ab) response to Tat, and some had cell-mediated immunity (CMI) as evaluated by skin test in vivo and T-cell proliferation in vitro. These results provide initial evidence of safety and potency of Tat toxoid vaccination in HIV-1-infected individuals.

  2. Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

    PubMed

    Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

    2017-10-01

    Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

    PubMed

    Berman, Joan W; Carvallo, Loreto; Buckner, Clarisa M; Luers, Aimée; Prevedel, Lisa; Bennett, Michael V; Eugenin, Eliseo A

    2016-03-02

    HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

  4. Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

    PubMed

    Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

    2012-11-02

    HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

  5. Functional characterization of a human cyclin T1 mutant reveals a different binding surface for Tat and HEXIM1.

    PubMed

    Kuzmina, Alona; Hadad, Uzi; Fujinaga, Koh; Taube, Ran

    2012-05-10

    HIV transcription is regulated at the step of elongation by the viral Tat protein and the cellular positive transcription elongation factor b (P-TEFb; Cdk9/cyclin T1). Herein, a human cyclin T1 mutant, cyclin T1-U7, which contains four substitutions and one deletion in the N-terminal cyclin box, was stably expressed in HeLa cells. HIV transcription was efficiently inhibited in HeLa-HA-CycT1-U7 stable cells. Cyclin T1-U7 bound Tat but did not modulate its expression levels, which remained high. Importantly cyclin T1-U7 failed to interact with Cdk9 or HEXIM1 and did not interfere with endogenous P-TEFb activity to stimulate MEF2C or NFkB mediated transcription. In a T cell line and primary CD4+ cells, cyclin T1-U7 also inhibited HIV transcription. We conclude that cyclin T1-U7 sequesters Tat from P-TEFb and inhibits HIV transcription. Importantly, N-terminal residues in cyclin T1 are specifically involved in the binding of cyclin T1 to HEXIM1 but not to Tat. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Synthesis and characterization of tat-mediated O-CMC magnetic nanoparticles having anticancer function

    NASA Astrophysics Data System (ADS)

    Zhao, Aijie; Yao, Peng; Kang, Chunshang; Yuan, Xubo; Chang, Jin; Pu, Peiyu

    2005-08-01

    This paper describes a new formulation of magnetic nanoparticles coated by a novel polymer matrix—O-carboxylmethylated chitosan (O-CMC) as drug/gene carrier. The O-CMC magnetic nanoparticles were derivatized with a peptide sequence from the HIV-tat protein to improve the translocational property and cellar uptake of the nanoparticles. To evaluate the O-MNPs-tat as drug carriers, MTX was incorporated as a model drug and MTX-loaded O-MNPs-tat with an average diameter of 45-60 nm were prepared and characterized by TEM, AFM and VSM. The cytotoxicity of MTX-loaded O-MNPs-tat was investigated with U-937 tumor cells. The results showed that the MTX-loaded O-MNPs-tat retained significant antitumor toxicity; additionally, sustained release of MTX from O-CMC nanoparticles was observed in vitro, suggesting that the tat-O-MNPs could be a novel magnetic targeting carrier.

  7. A lentiviral vector that activates latent human immunodeficiency virus-1 proviruses by the overexpression of tat and that kills the infected cells.

    PubMed

    Macías, David; Oya, Ricardo; Saniger, Luisa; Martín, Francisco; Luque, Francisco

    2009-11-01

    Despite the efficient HIV-1 replication blockage achieved with current highly active antiretroviral therapy (HAART) therapies, HIV-1 persists in the body and survives in a latent state that can last for the entire life of the patient. A long-lived reservoir of latently infected CD4(+) memory T cells represents the most important sanctuary for the virus and the greatest obstacle for viral eradication. In this work, we present an initial step toward a gene therapy approach aimed at the activation of latent provirus to induce the death of latently infected T cells. Latent HIV-1 infection is characterized by the failure of viral gene expression as a consequence of uninitiated or aborted transcription. We have constructed an HIV-1-based lentiviral vector (p5p53RTAT3) that expresses the viral trans-activating protein Tat in a drug-regulated manner and p53 in a Rev-dependent manner. We have demonstrated that the Tat-expressed protein from p5p53RTAT3 vector reactivates latent HIV-1 proviruses in J1.1 and ACH-2 cell lines and promotes p53-induced apoptosis in the presence of Rev. Our system was able to trigger the trans-activation of the provirus 5' long terminal repeat (LTR), stimulate the expression of the Rev protein from a tat-defective provirus, and provoke apoptosis selectively in the cells transfected with a tat-defective HIV-1 provirus in contrast to those with no HIV-1 provirus. However, the Rev-dependent p53 killing of latently infected cells was not effective enough for complete elimination of the awakened HIV-1 viruses. In summary, we have developed a vector system that is efficient in activating latent HIV-1 proviruses but that needs further improvement to kill infected cells.

  8. The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

    PubMed Central

    Lu, X; Welsh, T M; Peterlin, B M

    1993-01-01

    The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes. Images PMID:8445708

  9. Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

    PubMed

    Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki; Abe, Fumiyoshi

    2013-07-01

    Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

  10. Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

    PubMed Central

    Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki

    2013-01-01

    Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1K29R-K31R-GFP remained. The HPG1-1 (Rsp5P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure. PMID:23666621

  11. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

  12. The grafting of universal T-helper epitopes enhances immunogenicity of HIV-1 Tat concurrently improving its safety profile.

    PubMed

    Kashi, Venkatesh P; Jacob, Rajesh A; Shamanna, Raghavendra A; Menon, Malini; Balasiddaiah, Anangi; Varghese, Rebu K; Bachu, Mahesh; Ranga, Udaykumar

    2014-01-01

    Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

  13. Neuroimaging abnormalities in clade C HIV are independent of Tat genetic diversity.

    PubMed

    Paul, Robert H; Phillips, Sarah; Hoare, Jacqueline; Laidlaw, David H; Cabeen, Ryan; Olbricht, Gayla R; Su, Yuqing; Stein, Dan J; Engelbrecht, Susan; Seedat, Soraya; Salminen, Lauren E; Baker, Laurie M; Heaps, Jodi; Joska, John

    2017-04-01

    Controversy remains regarding the neurotoxicity of clade C human immunodeficiency virus (HIV-C). When examined in preclinical studies, a cysteine to serine substitution in the C31 dicysteine motif of the HIV-C Tat protein (C31S) results in less severe brain injury compared to other viral clades. By contrast, patient cohort studies identify significant neuropsychological impairment among HIV-C individuals independent of Tat variability. The present study clarified this discrepancy by examining neuroimaging markers of brain integrity among HIV-C individuals with and without the Tat substitution. Thirty-seven HIV-C individuals with the Tat C31S substitution, 109 HIV-C individuals without the Tat substitution (C31C), and 34 HIV- controls underwent 3T structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Volumes were determined for the caudate, putamen, thalamus, corpus callosum, total gray matter, and total white matter. DTI metrics included fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD). Tracts of interest included the anterior thalamic radiation (ATR), cingulum bundle (CING), uncinate fasciculus (UNC), and corpus callosum (CC). HIV+ individuals exhibited smaller volumes in subcortical gray matter, total gray matter and total white matter compared to HIV- controls. HIV+ individuals also exhibited DTI abnormalities across multiple tracts compared to HIV- controls. By contrast, neither volumetric nor diffusion indices differed significantly between the Tat C31S and C31C groups. Tat C31S status is not a sufficient biomarker of HIV-related brain integrity in patient populations. Clinical attention directed at brain health is warranted for all HIV+ individuals, independent of Tat C31S or clade C status.

  14. Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides.

    PubMed

    Strauch, Eva-Maria; Georgiou, George

    2007-11-23

    In vitro studies have suggested that the TatBC complex serves as the receptor for signal peptides targeted for export via the twin-arginine translocation (Tat) pathway. Substitution of the hallmark twin-arginine dipeptide with two lysines abrogates export of physiological substrates in all organisms. We report the isolation and characterization of suppressor mutations that allow export of an ssTor(KK)-GFP-SsrA tripartite fusion. We identified two amino acid suppressor mutations in the first cytoplasmic loop of TatC. In addition, two other amino acids in the first cytoplasmic loop exhibit epistatic suppression. Surprisingly, we also identified a suppressor mutation predicted to lie within the second periplasmic loop of TatC, a region that is not expected to interact directly with the signal peptide. The suppressor mutations allowed export of the native Esherichia coli Tat substrate trimethylamine N-oxide reductase with a twin-lysine substitution in its signal sequence. The cytoplasmic suppressor mutations conferred SDS sensitivity and partial filamentation, indicating that Tat export of authentic substrates was impaired.

  15. HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

    PubMed Central

    Col, Edwige; Caron, Cécile; Chable-Bessia, Christine; Legube, Gaelle; Gazzeri, Sylvie; Komatsu, Yasuhiko; Yoshida, Minoru; Benkirane, Monsef; Trouche, Didier; Khochbin, Saadi

    2005-01-01

    HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination. PMID:16001085

  16. Deciphering structure-activity relationships in a series of Tat/TAR inhibitors.

    PubMed

    Pascale, Lise; González, Alejandro López; Di Giorgio, Audrey; Gaysinski, Marc; Teixido Closa, Jordi; Tejedor, Roger Estrada; Azoulay, Stéphane; Patino, Nadia

    2016-11-01

    A series of pentameric "Polyamide Amino Acids" (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC50 ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.

  17. Caffeine Blocks HIV-1 Tat-Induced Amyloid Beta Production and Tau Phosphorylation.

    PubMed

    Soliman, Mahmoud L; Geiger, Jonathan D; Chen, Xuesong

    2017-03-01

    The increased life expectancy of people living with HIV-1 who are taking effective anti-retroviral therapeutics is now accompanied by increased Alzheimer's disease (AD)-like neurocognitive problems and neuropathological features such as increased levels of amyloid beta (Aβ) and phosphorylated tau proteins. Others and we have shown that HIV-1 Tat promotes the development of AD-like pathology. Indeed, HIV-1 Tat once endocytosed into neurons can alter morphological features and functions of endolysosomes as well as increase Aβ generation. Caffeine has been shown to have protective actions against AD and based on our recent findings that caffeine can inhibit endocytosis in neurons and can prevent neuronal Aβ generation, we tested the hypothesis that caffeine blocks HIV-1 Tat-induced Aβ generation and tau phosphorylation. In SH-SY5Y cells over-expressing wild-type amyloid beta precursor protein (AβPP), we demonstrated that HIV-1 Tat significantly increased secreted levels and intracellular levels of Aβ as well as cellular protein levels of phosphorylated tau. Caffeine significantly decreased levels of secreted and cellular levels of Aβ, and significantly blocked HIV-1 Tat-induced increases in secreted and cellular levels of Aβ. Caffeine also blocked HIV-1 Tat-induced increases in cellular levels of phosphorylated tau. Furthermore, caffeine blocked HIV-1 Tat-induced endolysosome dysfunction as indicated by decreased protein levels of vacuolar-ATPase and increased protein levels of cathepsin D. These results further implicate endolysosome dysfunction in the pathogenesis of AD and HAND, and by virtue of its ability to prevent and/or block neuropathological features associated with AD and HAND caffeine might find use as an effective adjunctive therapeutic agent.

  18. Trade-Space Analysis Tool for Constellations (TAT-C)

    NASA Technical Reports Server (NTRS)

    Le Moigne, Jacqueline; Dabney, Philip; de Weck, Olivier; Foreman, Veronica; Grogan, Paul; Holland, Matthew; Hughes, Steven; Nag, Sreeja

    2016-01-01

    Traditionally, space missions have relied on relatively large and monolithic satellites, but in the past few years, under a changing technological and economic environment, including instrument and spacecraft miniaturization, scalable launchers, secondary launches as well as hosted payloads, there is growing interest in implementing future NASA missions as Distributed Spacecraft Missions (DSM). The objective of our project is to provide a framework that facilitates DSM Pre-Phase A investigations and optimizes DSM designs with respect to a-priori Science goals. In this first version of our Trade-space Analysis Tool for Constellations (TAT-C), we are investigating questions such as: How many spacecraft should be included in the constellation? Which design has the best costrisk value? The main goals of TAT-C are to: Handle multiple spacecraft sharing a mission objective, from SmallSats up through flagships, Explore the variables trade space for pre-defined science, cost and risk goals, and pre-defined metrics Optimize cost and performance across multiple instruments and platforms vs. one at a time.This paper describes the overall architecture of TAT-C including: a User Interface (UI) interacting with multiple users - scientists, missions designers or program managers; an Executive Driver gathering requirements from UI, then formulating Trade-space Search Requests for the Trade-space Search Iterator first with inputs from the Knowledge Base, then, in collaboration with the Orbit Coverage, Reduction Metrics, and Cost Risk modules, generating multiple potential architectures and their associated characteristics. TAT-C leverages the use of the Goddard Mission Analysis Tool (GMAT) to compute coverage and ancillary data, streamlining the computations by modeling orbits in a way that balances accuracy and performance.TAT-C current version includes uniform Walker constellations as well as Ad-Hoc constellations, and its cost model represents an aggregate model consisting of

  19. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

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

    Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides formore » calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein

  20. Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine.

    PubMed

    Zagury, J F; Sill, A; Blattner, W; Lachgar, A; Le Buanec, H; Richardson, M; Rappaport, J; Hendel, H; Bizzini, B; Gringeri, A; Carcagno, M; Criscuolo, M; Burny, A; Gallo, R C; Zagury, D

    1998-01-01

    To investigate which immune parameters, such as antibodies against HIV-1 specificities, or viral parameters, such as p24 antigenemia, are predictive of disease progression. We performed studies on serum collected from individuals exhibiting two extremes of disease evolution--67 fast progressors (FP) and 182 nonprogressors (NP)--at their enrollment. After a 1- to 2-year clinical follow-up of 104 nonprogressors after their enrollment, we could determine the best serologic predictors for disease progression. We investigated levels of antibodies to tetanus toxoid and to HIV antigens including Env, Gag, Nef, and Tat proteins, as well as p24 antigenemia, viremia, CD4 cell count, and interferon-alpha (IFN-alpha) titers in FPs and NPs, and we correlated these data with clinical and biologic signs of progression. p24 Antigenemia, a marker of viral replication, and anti-Tat antibodies were highly and inversely correlated in both groups (P < .001). Furthermore, anti-p24 antibodies and low serum IFN-alpha levels were correlated to the NP versus the FP cohort. Finally, among NPs, only antibodies to Tat and not to the other HIV specificities (Env, Nef, Gag) were significantly predictive of clinical stability during their follow-up. Antibodies toward HIV-1 Tat, which are inversely correlated to p24 antigenemia, appear as a critical marker for a lack of disease progression. This study strongly suggests that rising anti-Tat antibodies through active immunization may be beneficial in AIDS vaccine development to control viral replication.

  1. HIV-1 Tat protein promotes formation of more-processive elongation complexes.

    PubMed Central

    Marciniak, R A; Sharp, P A

    1991-01-01

    The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

  2. Intelligent tit-for-tat in the iterated prisoner's dilemma game

    NASA Astrophysics Data System (ADS)

    Baek, Seung Ki; Kim, Beom Jun

    2008-07-01

    We seek a route to the equilibrium where all the agents cooperate in the iterated prisoner’s dilemma game on a two-dimensional plane, focusing on the role of tit-for-tat strategy. When a time horizon, within which a strategy can recall the past, is one time step, an equilibrium can be achieved as cooperating strategies dominate the whole population via proliferation of tit-for-tat. Extending the time horizon, we filter out poor strategies by simplified replicator dynamics and observe a similar evolutionary pattern to reach the cooperating equilibrium. In particular, the rise of a modified tit-for-tat strategy plays a central role, which implies how a robust strategy is adopted when provided with an enhanced memory capacity.

  3. HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

    PubMed

    Kesby, James P; Najera, Julia A; Romoli, Benedetto; Fang, Yiding; Basova, Liana; Birmingham, Amanda; Marcondes, Maria Cecilia G; Dulcis, Davide; Semenova, Svetlana

    2017-10-01

    Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for

  4. Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

    PubMed Central

    Gruber, Andreas; Lell, Claudia P; Speth, Cornelia; Stoiber, Heribert; Lass-Flörl, Cornelia; Sonneborn, Anja; Ernst, Joachim F; Dierich, Manfred P; Würzner, Reinhard

    2001-01-01

    Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis. PMID:11899432

  5. Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Hiraki, Toshiki; Usui, Keiko; Abe, Fumiyoshi

    2010-12-01

    Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp-) strains under pressures of 15-25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp- cells to grow at 15-25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

  6. [Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

    PubMed

    Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

    2013-05-04

    To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

  7. Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant.

    PubMed

    Watkins, Jennifer D; Campbell, Grant R; Halimi, Hubert; Loret, Erwann P

    2008-09-22

    The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70 degrees C showed that Tat Eli is not a random coil at 20 degrees C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes.

  8. Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y.

    PubMed

    Choi, Sunghyun; Oh, Jae Hoon; Kim, Hyeseon; Nam, So Hee; Shin, Jeehae; Park, Jong-Sang

    2015-10-01

    Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.

  9. Twin-arginine signal peptide of Bacillus subtilis YwbN can direct Tat-dependent secretion of methyl parathion hydrolase.

    PubMed

    Liu, Ruihua; Zuo, Zhenqiang; Xu, Yingming; Song, Cunjiang; Jiang, Hong; Qiao, Chuanling; Xu, Ping; Zhou, Qixing; Yang, Chao

    2014-04-02

    The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

  10. Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans.

    PubMed

    Valverde, José R; Gullón, Sonia; Mellado, Rafael P

    2018-06-14

    Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and

  11. Covalent attachment of TAT peptides and thiolated alkyl molecules on GaAs surfaces.

    PubMed

    Cho, Youngnam; Ivanisevic, Albena

    2005-07-07

    Four TAT peptide fragments were used to functionalize GaAs surfaces by adsorption from solution. In addition, two well-studied alkylthiols, mercaptohexadecanoic acid (MHA) and 1-octadecanethiol (ODT) were utilized as references to understand the structure of the TAT peptide monolayer on GaAs. The different sequences of TAT peptides were employed in recognition experiments where a synthetic RNA sequence was tested to verify the specific interaction with the TAT peptide. The modified GaAs surfaces were characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS). AFM studies were used to compare the surface roughness before and after functionalization. XPS allowed us to characterize the chemical composition of the GaAs surface and conclude that the monolayers composed of different sequences of peptides have similar surface chemistries. Finally, FT-IRRAS experiments enabled us to deduce that the TAT peptide monolayers have a fairly ordered and densely packed alkyl chain structure. The recognition experiments showed preferred interaction of the RNA sequence toward peptides with high arginine content.

  12. Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant

    PubMed Central

    Watkins, Jennifer D; Campbell, Grant R; Halimi, Hubert; Loret, Erwann P

    2008-01-01

    Background The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. Results Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70°C showed that Tat Eli is not a random coil at 20°C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. Conclusion We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes. PMID:18808674

  13. Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant.

    PubMed

    Borsutzky, Stefan; Fiorelli, Valeria; Ebensen, Thomas; Tripiciano, Antonella; Rharbaoui, Faiza; Scoglio, Arianna; Link, Claudia; Nappi, Filomena; Morr, Michael; Buttó, Stefano; Cafaro, Aurelio; Mühlradt, Peter F; Ensoli, Barbara; Guzmán, Carlos A

    2003-06-01

    A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

  14. Transduced Tat-DJ-1 Protein Protects against Oxidative Stress-Induced SH-SY5Y Cell Death and Parkinson Disease in a Mouse Model

    PubMed Central

    Jeong, Hoon Jae; Kim, Dae Won; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Park, Meeyoung; Kim, Duk-Soo; Kwon, Oh-Shin; Hwang, In Koo; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2012-01-01

    Parkinson’s disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD. PMID:22526393

  15. Enquête internationale sur l'état de l'art et l'état de la pratique en géotechnique

    NASA Astrophysics Data System (ADS)

    Acosta-Martinez, Hugo; Delage, Pierre; Nicks, Jennifer; Day, Peter

    2018-05-01

    Cet article présente une synthèse des résultats de l'enquête internationale sur l'état de l'art et l'état de la pratique en ingénierie géotechnique lancée par le Groupe présidentiel des entreprises associées et le Comité de supervision technique de la Société internationale de mécanique des sols et de géotechnique en mars 2017. Il résume également les discussions qui ont eu lieu sur le sujet durant le 19e CIMSG à Séoul, le 20 septembre 2017.

  16. Molecular Dynamics Simulation and Experimental Verification of the Interaction between Cyclin T1 and HIV-1 Tat Proteins

    PubMed Central

    Asamitsu, Kaori; Hibi, Yurina

    2015-01-01

    The viral encoded Tat protein is essential for the transcriptional activation of HIV proviral DNA. Interaction of Tat with a cellular transcription elongation factor P-TEFb containing CycT1 is critically required for its action. In this study, we performed MD simulation using the 3D data for wild-type and 4CycT1mutants3D data. We found that the dynamic structural change of CycT1 H2’ helix is indispensable for its activity for the Tat action. Moreover, we detected flexible structural changes of the Tat-recognition cavity in the WT CycT1 comprising of ten AAs that are in contact with Tat. These structural fluctuations in WT were lost in the CycT1 mutants. We also found the critical importance of the hydrogen bond network involving H1, H1’ and H2 helices of CycT1. Since similar AA substitutions of the Tat-CycT1 chimera retained the Tat-supporting activity, these interactions are considered primarily involved in interaction with Tat. These findings described in this paper should provide vital information for the development of effective anti-Tat compound. PMID:25781978

  17. Effects of human chromosome 12 on interactions between Tat and TAR of human immunodeficiency virus type 1.

    PubMed Central

    Alonso, A; Cujec, T P; Peterlin, B M

    1994-01-01

    Rates of transcriptions of the human immunodeficiency virus are greatly increased by the viral trans activator Tat. In vitro, Tat binds to the 5' bulge of the trans-activation response (TAR) RNA stem-loop, which is present in all viral transcripts. In human cells, the central loop in TAR and its cellular RNA-binding proteins are also critical for the function of Tat. Previously, we demonstrated that in rodent cells (CHO cells), but not in those which contain the human chromosome 12 (CHO12 cells), Tat-TAR interactions are compromised. In this study, we examined the roles of the bulge and loop in TAR in Tat trans activation in these cells. Whereas low levels of trans activation depended solely on interactions between Tat and the bulge in CHO cells, high levels of trans activation depended also on interactions between Tat and the loop in CHO12 cells. Since the TAR loop binding proteins in these two cell lines were identical and different from their human counterpart, the human chromosome 12 does not encode TAR loop binding proteins. In vivo binding competition studies with TAR decoys confirmed that the binding of Tat to TAR is more efficient in CHO12 cells. Thus, the protein(s) encoded on human chromosome 12 helps to tether Tat to TAR via its loop, which results in high levels of trans activation. Images PMID:8083988

  18. Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

    PubMed

    Ronsard, Larance; Ganguli, Nilanjana; Singh, Vivek K; Mohankumar, Kumaravel; Rai, Tripti; Sridharan, Subhashree; Pajaniradje, Sankar; Kumar, Binod; Rai, Devesh; Chaudhuri, Suhnrita; Coumar, Mohane S; Ramachandran, Vishnampettai G; Banerjea, Akhil C

    2017-01-01

    HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans -activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans -activation response (TAR) RNA. In this study, HIV-1 infected patients ( n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

  19. The effects of HIV-1 regulatory TAT protein expression on brain reward function, response to psychostimulants and delay-dependent memory in mice.

    PubMed

    Kesby, James P; Markou, Athina; Semenova, Svetlana

    2016-10-01

    Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

    2006-02-01

    Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

  1. Defining the Pathway for Tat-mediated Delivery of β-Glucuronidase in Cultured Cells and MPS VII Mice

    PubMed Central

    Orii, Koji O.; Grubb, Jeffrey H.; Vogler, Carole; Levy, Beth; Tan, Yun; Markova, Kamelia; Davidson, Beverly L.; Mao, Q.; Orii, Tadao; Kondo, Naomi; Sly, William S.

    2008-01-01

    We used recombinant forms of human β-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS-Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS-Tat fusion protein showed only 30-50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS-Tat was less rapidly cleared from the circulation in the rat (t1/2 = 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS-Tat was more efficiently delivered to kidney. Histology showed that GUS-Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme. PMID:16043103

  2. Application of Bacillus sp. TAT105 to reduce ammonia emissions during pilot-scale composting of swine manure.

    PubMed

    Kuroda, Kazutaka; Tanaka, Akihiro; Furuhashi, Kenich; Nakasaki, Kiyohiko

    2017-12-01

    Thermophilic ammonium-tolerant bacterium Bacillus sp. TAT105 grows and reduces ammonia (NH 3 ) emissions by assimilating ammonium nitrogen during composting of swine feces. To evaluate the efficacy of a biological additive containing TAT105 at reducing NH 3 emissions, composting tests of swine manure on a pilot scale (1.8 m 3 ) were conducted. In the TAT105-added treatment, NH 3 emissions and nitrogen loss were lower than those in the control treatment without TAT105. No significant difference was detected in losses in the weight and volatile solids between the treatments. Concentration of thermophilic ammonium-tolerant bacteria in the compost increased in both treatments at the initial stage of composting. In the TAT105-added treatment, bacterial concentration reached ~10 9 colony-forming units per gram of dry matter, several-fold higher than that in the control and stayed at the same level until the end. These results suggest that TAT105 grows during composting and reduces NH 3 emissions in TAT105-added treatment.

  3. Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up.

    PubMed

    Longo, Olimpia; Tripiciano, Antonella; Fiorelli, Valeria; Bellino, Stefania; Scoglio, Arianna; Collacchi, Barbara; Alvarez, Maria Josè Ruiz; Francavilla, Vittorio; Arancio, Angela; Paniccia, Giovanni; Lazzarin, Adriano; Tambussi, Giuseppe; Din, Chiara Tassan; Visintini, Raffaele; Narciso, Pasquale; Antinori, Andrea; D'Offizi, Gianpiero; Giulianelli, Marina; Carta, Maria; Di Carlo, Aldo; Palamara, Guido; Giuliani, Massimo; Laguardia, Maria Elena; Monini, Paolo; Magnani, Mauro; Ensoli, Fabrizio; Ensoli, Barbara

    2009-05-26

    A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

  4. Tat-functionalized liposomes for the treatment of meningitis: an in vitro study

    PubMed Central

    Bartomeu Garcia, Caterina; Shi, Di; Webster, Thomas J

    2017-01-01

    Bacterial meningitis has become a global concern, because of the emergence of antibiotic-resistant bacteria. It has been demonstrated that liposomes can enter bacteria, thus providing a possible treatment for numerous infections, including meningitis. Fusogenic liposomes are pH-sensitive with a high capacity to fuse with the bacteria membrane and promote intracellular drug release. Moreover, this ability can be improved by using cell-penetrating peptides (such as Tat47–57, which is a peptide derived from the Tat protein of HIV). The purpose of this in vitro study was to demonstrate for the first time the ability of the presently prepared fusogenic liposomes, which were spherical particles with a diameter of 100 nm loaded with antibiotics and functionalized with-cell penetrating peptides (Tat47–57), to fight the main bacteria that cause meningitis. For this, vancomycin, methicillin, and ampicillin antibiotics were loaded inside fusogenic liposomes to fight Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, and Escherichia coli. Antibacterial activity of Tat-functionalized and nonfunctionalized liposomes loaded with antibiotics was tested by determining bacteria colony-forming units and growth-curve assays coupled with live/dead assays using fluorescence microscopy. Results showed a remarkable decrease in antibiotic minimum inhibitory concentration when all of the bacteria were treated with these novel liposomes, especially for the functionalized liposomes loaded with methicillin. With antibiotic concentrations of 1.7–3 µg/mL for Tat-functionalized liposomes loaded with methicillin, the bacteria population was totally eradicated. Cytotoxicity tests with astrocytes and endothelial cells, major cellular components of the blood–brain barrier, were also performed for all of the liposomes, including free antibiotic and the Tat peptide. Results showed much promise for the further study of the presently formulated liposomes to treat meningitis

  5. In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

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

    Barbon, Silvia, E-mail: silvia.barbon@yahoo.it

    In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in themore » future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT

  6. Parallel conduction of the phase I preventive and therapeutic trials based on the Tat vaccine candidate.

    PubMed

    Bellino, S; Francavilla, V; Longo, O; Tripiciano, A; Paniccia, G; Arancio, A; Fiorelli, V; Scoglio, A; Collacchi, B; Campagna, M; Lazzarin, A; Tambussi, G; Din, C Tassan; Visintini, R; Narciso, P; Antinori, A; D'Offizi, G; Giulianelli, M; Carta, M; Di Carlo, A; Palamara, G; Giuliani, M; Laguardia, M E; Monini, P; Magnani, M; Ensoli, F; Ensoli, B

    2009-09-01

    The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug

  7. The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency

    PubMed Central

    Mousseau, Guillaume; Kessing, Cari F.; Fromentin, Rémi; Trautmann, Lydie; Chomont, Nicolas

    2015-01-01

    ABSTRACT Antiretroviral therapy (ART) inhibits HIV-1 replication, but the virus persists in latently infected resting memory CD4+ T cells susceptible to viral reactivation. The virus-encoded early gene product Tat activates transcription of the viral genome and promotes exponential viral production. Here we show that the Tat inhibitor didehydro-cortistatin A (dCA), unlike other antiretrovirals, reduces residual levels of viral transcription in several models of HIV latency, breaks the Tat-mediated transcriptional feedback loop, and establishes a nearly permanent state of latency, which greatly diminishes the capacity for virus reactivation. Importantly, treatment with dCA induces inactivation of viral transcription even after its removal, suggesting that the HIV promoter is epigenetically repressed. Critically, dCA inhibits viral reactivation upon CD3/CD28 or prostratin stimulation of latently infected CD4+ T cells from HIV-infected subjects receiving suppressive ART. Our results suggest that inclusion of a Tat inhibitor in current ART regimens may contribute to a functional HIV-1 cure by reducing low-level viremia and preventing viral reactivation from latent reservoirs. PMID:26152583

  8. Not Your Same Old Story: New Rules for Thematic Apperceptive Techniques (TATs).

    PubMed

    Jenkins, Sharon Rae

    2017-01-01

    Stories told about pictures have been used for both research and clinical practice since the beginning of modern personality assessment. However, with the growing science-practice gap, these thematic apperceptive techniques (TATs) have been used differently in those 2 venues. Scientific validation is presumptively general, but clinical application is idiographic and situation-specific. A bridge is needed. The manualized human-scored narrative analysis systems discussed here are valuable scientist-practitioner tools, but they require a validation literature to support further research publication, maintain their role in clinical training, and justify clinicians' reimbursement by third-party payers. To facilitate wider understanding of manualized TAT methodologies, this article addresses long-standing criticisms of TAT reliability and proposes some strategic solutions to the measurement error problem for both researchers and clinicians, including analyzing person-situation interactions, purposeful situation sampling for within-storyteller comparisons, and uses of small samples. The new rules for TATs include conceptual and methodological standards that researchers should aim to meet and report, reviewers should apply to manuscripts, and clinical assessors can use to analyze their own data and justify third-party payment.

  9. TIT FOR TAT in sticklebacks and the evolution of cooperation

    NASA Astrophysics Data System (ADS)

    Milinski, Manfred

    1987-01-01

    The problems of achieving mutual cooperation can be formalized in a game called the Prisoner's Dilemma in which selfish defection is always more rewarding than cooperation1. If the two protagonists have a certain minimum probability of meeting again a strategy called TIT FOR TAT is very successful2. In TIT FOR TAT the player cooperates on the first move and thereafter does whatever the opponent did on the previous move. I have studied the behaviour of fish when confronting a potential predator, because conflicts can arise within pairs of fish in these circumstances which I argue resemble a series of games of Prisoner's Dilemma. Using a system of mirrors, single three-spined sticklebacks (Gasterosteus aculeatus) approaching a live predator were provided with either a simulated cooperating companion or a simulated defecting one. In both cases the test fish behaved according to TIT FOR TAT supporting the hypothesis that cooperation can evolve among egoists.

  10. Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

    PubMed Central

    Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

    2013-01-01

    Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial

  11. Immune Responses of HIV-1 Tat Transgenic Mice to Mycobacterium Tuberculosis W-Beijing SA161

    PubMed Central

    Honda, Jennifer R; Shang, Shaobin; Shanley, Crystal A; Caraway, Megan L; Henao-Tamayo, Marcela; Chan, Edward D; Basaraba, Randall J; Orme, Ian M; Ordway, Diane J; Flores, Sonia C

    2011-01-01

    Background: Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis. Results: Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijing SA161. Survival assays demonstrate accelerated mortality rates of the Tat transgenic mice compared to non-transgenics. Tat transgenic mice also showed poorly organized lung granulomata-like lesions. Analysis of the host immune response using quantitative RT-PCR, flow cytometry for surface markers, and intracellular cytokine staining showed increased expression of pro-inflammatory cytokines in the lungs, increased numbers of cells expressing ICAM1, increased numbers of CD4+CD25+Foxp3+ T regulatory cells, and IL-4 producing CD4+ T cells in the Tat transgenics compared to infected non-tg mice. Conclusions: Our data show quantitative differences in the inflammatory response to the SA161 clinical isolate of M. tuberculosis W-Beijing between Tat transgenic and non-transgenic mice, suggesting Tat contributes to the pathogenesis of tuberculosis. PMID:22046211

  12. The Relationship Between Behavioral Indices of Aggression and Hostile Content on the TAT

    ERIC Educational Resources Information Center

    Matranga, James T.

    1976-01-01

    Adolescent male delinquents were administered the Thematic Apperception Test (TAT) to examine the relationship between behavioral indices of aggression and hostility. The results of this investigation supported the hypothesis that an inverse relationship exists between hostility on the TAT and ratings of aggressive behavior in adolescent males.…

  13. TAT-Mediated Delivery of Tousled Protein to Salivary Glands Protects Against Radiation-Induced Hypofunction

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

    Sunavala-Dossabhoy, Gulshan, E-mail: gsunav@lsuhsc.edu; Palaniyandi, Senthilnathan; Richardson, Charles

    2012-09-01

    Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect againstmore » IR-induced salivary hypofunction. Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D{sub 0} = 4.13 {+-} 1.0 Gy; {alpha}/{beta} = 0 Gy) compared with cells transduced with the TAT peptide (D{sub 0} = 4.91 {+-} 1.0 Gy; {alpha}/{beta} = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.« less

  14. Business Case Analysis: Continuous Integrated Logistics Support-Targeted Allowance Technique (CILS-TAT)

    DTIC Science & Technology

    2013-06-01

    In this research, we examine the Naval Sea Logistics Command s Continuous Integrated Logistics Support Targeted Allowancing Technique (CILS TAT) and... the feasibility of program re-implementation. We conduct an analysis of this allowancing method s effectiveness onboard U.S. Navy Ballistic Missile...Defense (BMD) ships, measure the costs associated with performing a CILS TAT, and provide recommendations concerning possible improvements to the

  15. HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

    PubMed

    Vivarini, Áislan de Carvalho; Pereira, Renata de Meirelles Santos; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C; Saraiva, Elvira M; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

    2015-11-26

    HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

  16. Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles

    PubMed Central

    Peetla, Chiranjeevi; Rao, Kavitha S.; Labhasetwar, Vinod

    2009-01-01

    The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly (L-lactide) nanoparticles (RNPs), either conjugated to a TAT peptide (TAT-RNPs) or scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (π–A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir–Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP–membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift towards higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM whereas RNPs were seen anchored loosely to the

  17. Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT

    NASA Astrophysics Data System (ADS)

    Hu, Shanshan; Wang, Tong; Pei, Xibo; Cai, He; Chen, Junyu; Zhang, Xin; Wan, Qianbing; Wang, Jian

    2016-10-01

    In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

  18. The Narrative Arc of TATs: Introduction to the JPA Special Section on Thematic Apperceptive Techniques.

    PubMed

    Jenkins, Sharon Rae

    2017-01-01

    The past decade has seen important developments in thematic apperceptive techniques (TATs), with the creation of new card sets having alternate pictures representing different cultures, new scoring systems becoming available, and increasing international communication of these achievements. However, continuing impediments to the development of a validational literature include lingering mistaken assumptions about the nature of story data, ongoing debates about appropriate psychometric evaluation, and continuing questions about how stimuli and scoring systems should be conceptualized and interpreted. Negotiating the publication system can impede some potential authors. Excellent work on TATs with children is not well known in the adult-focused journals. The labor burden of meeting increasingly sophisticated publication standards might be a barrier to assessors focused on clinical practice. Accumulating a focused evidence base is challenging given the diversity of criterion variables for which TATs have been used. Research on TATs by clinicians can span the science-practice gap, but the narrative arc can be a dramatic one. The articles in this special section on TATs represent important conceptual, methodological, and substantive innovations.

  19. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  20. Business Case Analysis: Continuous Integrated Logistics Support-Targeted Allowance Technique (CILS-TAT)

    DTIC Science & Technology

    2013-05-30

    In this research, we examine the Naval Sea Logistics Command’s Continuous Integrated Logistics Support-Targeted Allowancing Technique (CILS-TAT) and... the feasibility of program re-implementation. We conduct an analysis of this allowancing method’s effectiveness onboard U.S. Navy Ballistic Missile...Defense (BMD) ships, measure the costs associated with performing a CILS-TAT, and provide recommendations concerning possible improvements to the

  1. TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors.

    PubMed

    Sethuraman, Vijay A; Bae, You Han

    2007-04-02

    A novel drug targeting system for acidic solid tumors has been developed based on ultra pH-sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of poly(l-lactic acid) (PLLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to TAT (TAT micelle), 2) an ultra pH-sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TAT micelles had particle sizes between 20 and 45 nm and their critical micelle concentrations were 3.5 mg/l to 5.5 mg/l. The TAT micelles, upon mixing with pH-sensitive PSD-b-PEG, showed a slight increase in particle size between pH 8.0 and 6.8 (60-90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flow cytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The confocal microscopy indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above micelles would be able to target any hydrophobic drug near the nucleus.

  2. A Role for p38 Mitogen-activated Protein Kinase-mediated Threonine 30-dependent Norepinephrine Transporter Regulation in Cocaine Sensitization and Conditioned Place Preference*

    PubMed Central

    Mannangatti, Padmanabhan; NarasimhaNaidu, Kamalakkannan; Damaj, Mohamad Imad; Ramamoorthy, Sammanda; Jayanthi, Lankupalle Damodara

    2015-01-01

    The noradrenergic and p38 mitogen-activated protein kinase (p38 MAPK) systems are implicated in cocaine-elicited behaviors. Previously, we demonstrated a role for p38 MAPK-mediated norepinephrine transporter (NET) Thr30 phosphorylation in cocaine-induced NET up-regulation (Mannangatti, P., Arapulisamy, O., Shippenberg, T. S., Ramamoorthy, S., and Jayanthi, L. D. (2011) J. Biol. Chem. 286, 20239–20250). The present study explored the functional interaction between p38 MAPK-mediated NET regulation and cocaine-induced behaviors. In vitro cocaine treatment of mouse prefrontal cortex synaptosomes resulted in enhanced NET function, surface expression, and phosphorylation. Pretreatment with PD169316, a p38 MAPK inhibitor, completely blocked cocaine-mediated NET up-regulation and phosphorylation. In mice, in vivo administration of p38 MAPK inhibitor SB203580 completely blocked cocaine-induced NET up-regulation and p38 MAPK activation in the prefrontal cortex and nucleus accumbens. When tested for cocaine-induced locomotor sensitization and conditioned place preference (CPP), mice receiving SB203580 on cocaine challenge day or on postconditioning test day exhibited significantly reduced cocaine sensitization and CPP. A transactivator of transcription (TAT) peptide strategy was utilized to test the involvement of the NET-Thr30 motif. In vitro treatment of synaptosomes with TAT-NET-Thr30 (wild-type peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In vivo administration of TAT-NET-Thr30 peptide but not TAT-NET-T30A (mutant peptide) completely blocked cocaine-mediated NET up-regulation and phosphorylation. In the cocaine CPP paradigm, mice receiving TAT-NET-Thr30 but not TAT-NET-T30A on postconditioning test day exhibited significantly reduced cocaine CPP. Following extinction, TAT-NET-Thr30 when given prior to cocaine challenge significantly reduced reinstatement of cocaine CPP. These results demonstrate that the direct inhibition of p38

  3. Assisted annotation of medical free text using RapTAT

    PubMed Central

    Gobbel, Glenn T; Garvin, Jennifer; Reeves, Ruth; Cronin, Robert M; Heavirland, Julia; Williams, Jenifer; Weaver, Allison; Jayaramaraja, Shrimalini; Giuse, Dario; Speroff, Theodore; Brown, Steven H; Xu, Hua; Matheny, Michael E

    2014-01-01

    Objective To determine whether assisted annotation using interactive training can reduce the time required to annotate a clinical document corpus without introducing bias. Materials and methods A tool, RapTAT, was designed to assist annotation by iteratively pre-annotating probable phrases of interest within a document, presenting the annotations to a reviewer for correction, and then using the corrected annotations for further machine learning-based training before pre-annotating subsequent documents. Annotators reviewed 404 clinical notes either manually or using RapTAT assistance for concepts related to quality of care during heart failure treatment. Notes were divided into 20 batches of 19–21 documents for iterative annotation and training. Results The number of correct RapTAT pre-annotations increased significantly and annotation time per batch decreased by ∼50% over the course of annotation. Annotation rate increased from batch to batch for assisted but not manual reviewers. Pre-annotation F-measure increased from 0.5 to 0.6 to >0.80 (relative to both assisted reviewer and reference annotations) over the first three batches and more slowly thereafter. Overall inter-annotator agreement was significantly higher between RapTAT-assisted reviewers (0.89) than between manual reviewers (0.85). Discussion The tool reduced workload by decreasing the number of annotations needing to be added and helping reviewers to annotate at an increased rate. Agreement between the pre-annotations and reference standard, and agreement between the pre-annotations and assisted annotations, were similar throughout the annotation process, which suggests that pre-annotation did not introduce bias. Conclusions Pre-annotations generated by a tool capable of interactive training can reduce the time required to create an annotated document corpus by up to 50%. PMID:24431336

  4. Ketone bodies protection against HIV-1 Tat-induced neurotoxicity.

    PubMed

    Hui, Liang; Chen, Xuesong; Bhatt, Dhaval; Geiger, Nicholas H; Rosenberger, Thad A; Haughey, Norman J; Masino, Susan A; Geiger, Jonathan D

    2012-07-01

    HIV-1-associated neurocognitive disorder (HAND) is a syndrome that ranges clinically from subtle neuropsychological impairments to profoundly disabling HIV-associated dementia. Not only is the pathogenesis of HAND unclear, but also effective treatments are unavailable. The HIV-1 transactivator of transcription protein (HIV-1 Tat) is strongly implicated in the pathogenesis of HAND, in part, because of its well-characterized ability to directly excite neurons and cause neurotoxicity. Consistent with previous findings from others, we demonstrate here that HIV-1 Tat induced neurotoxicity, increased intracellular calcium, and disrupted a variety of mitochondria functions, such as reducing mitochondrial membrane potential, increasing levels of reactive oxygen species, and decreasing bioenergetic efficiency. Of therapeutic importance, we show that treatment of cultured neurons with ketone bodies normalized HIV-1 Tat induced changes in levels of intracellular calcium, mitochondrial function, and neuronal cell death. Ketone bodies are normally produced in the body and serve as alternative energy substrates in tissues including brain and can cross the blood-brain barrier. Ketogenic strategies have been used clinically for treatment of neurological disorders and our current results suggest that similar strategies may also provide clinical benefits in the treatment of HAND. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  5. Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

    PubMed Central

    Roy, Chandra Nath; Promjunyakul, Warunya; Hatakeyama, Hiroyasu; Gonda, Kohsuke; Imamura, Junji; Vasudevanpillai, Biju; Ohuchi, Noriaki; Kanzaki, Makoto; Higuchi, Hideo; Kaku, Mitsuo

    2013-01-01

    The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines. PMID:23732912

  6. The HIV-1 viral protein Tat increases glutamate and decreases GABA exocytosis from human and mouse neocortical nerve endings.

    PubMed

    Musante, Veronica; Summa, Maria; Neri, Elisa; Puliti, Aldamaria; Godowicz, Tomasz T; Severi, Paolo; Battaglia, Giuseppe; Raiteri, Maurizio; Pittaluga, Anna

    2010-08-01

    Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.

  7. Oral Administration of TAT-PTD-Diapause Hormone Fusion Protein Interferes With Helicoverpa armigera (Lepidoptera: Noctuidae) Development.

    PubMed

    Zhou, Zhou; Li, Yongli; Yuan, Chunyan; Zhang, Yongan; Qu, Liangjian

    2015-01-01

    Diapause hormone (DH), which can terminate diapause in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), has shown promise as a pest control method. However, the main challenge in using DH as an insecticide lies in achieving effective oral delivery, since the peptide may be degraded by digestive enzymes in the gut. To improve the efficacy of oral DH application, the Clostera anastomosis (L.) (Lepidoptera: Notodontidae) diapause hormone (caDH) was fused to the Protein Transduction Domain (PTD) of the human immunodeficiency virus-1 transactivator of transcription (TAT). Cellular transduction of TAT-caDH was verified with the use of a green fluorescent protein fusion, and its ability to terminate diapause was verified by injection into diapausing H. armigera pupae. Orally administered TAT-caDH resulted in larval growth inhibition. In TAT-caDH-treated insects, larval duration was delayed and the pupation rates were decreased at both development promoting conditions [27 °C, a photoperiod of 14:10(L:D) h] and diapause inducing conditions [20 °C, a photoperiod of 10:14(L:D) h]. No significant difference in diapause rate was observed between the TAT-caDH-treated and caDH-treated or control pupae maintained at diapause inducing conditions. Our results show that treatment with a recombinant TAT-caDH protein can affect larval development in H. armigera, and it suggest that TAT-DH treatment may be useful for controlling pests. This study is the first record of oral DH application in insect. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

  8. Chimeric Peptide Tat-HA-NR2B9c Improves Regenerative Repair after Transient Global Ischemia.

    PubMed

    Zhou, Hai-Hui; Zhang, Li; Zhang, Hai-Xia; Zhang, Jin-Ping; Ge, Wei-Hong

    2017-01-01

    Transient global ischemia (TGI) is a major public health problem, and it heightens the need of effective treatments. The present study was undertaken to investigate whether recombinant polypeptide Tat-HA-NR2B9c improves spatial learning and memory deficits in rats after TGI. Rats were subjected to 20-min ischemia induced by four-vessel occlusion (4-VO) method and daily injected with Tat-HA-NR2B9c (1.12 mg/kg) for 1 week. Tat-HA-NR2B9c increased CREB activity, upregulated B-cell lymphoma-2 (Bcl-2) expression after treated for 24 h. There was a significant increase in dendrite spine density in hippocampal CA1 region and BrdU-positive cells and BrdU/NeuN-positive cells in the dentate gyrus after Tat-HA-NR2B9c treatment, compared with ischemia group at postischemic day 28. Inhibition of the CREB activation by recombinant lentivirus, LV-CREB133-GFP, abolished the upregulation effects of Tat-HA-NR2B9c on Bcl-2 expression. Moreover, Tat-HA-NR2B9c improved the impaired spatial learning and memory ability in Morris water maze. These results suggest that Tat-HA-NR2B9c substantially ameliorated the TGI-induced loss of dendrite spine in hippocampal CA1, increased neurogenesis in dentate gyrus, and significantly improved cognitive abilities by the CREB pathway in rats after transient global cerebral ischemia. It may be served as a treatment for TGI.

  9. Eudragit S100-Coated Chitosan Nanoparticles Co-loading Tat for Enhanced Oral Colon Absorption of Insulin.

    PubMed

    Chen, Shuangxi; Guo, Feng; Deng, Tiantian; Zhu, Siqi; Liu, Wenyu; Zhong, Haijun; Yu, Hua; Luo, Rong; Deng, Zeyuan

    2017-05-01

    In order to improve oral absorption of insulin, especially the absorption at the colon, Eudragit S100® (ES)-coated chitosan nanoparticles loading insulin and a trans-activating transcriptional peptide (Tat) were employed as the vehicle. In vitro releases of insulin and Tat from ES-coated chitosan nanoparticles had a pH-dependant characteristic. A small amount of the contents was released from the coated nanoparticles at pH 1.2 simulated gastric fluid, while a fairly fast and complete release was observed in pH 7.4 medium. Caco-2 cell was used as the model of cellular transport and uptake studies. The results showed that the cellular transport and uptake of insulin for ES-coated chitosan nanoparticles co-loading insulin and Tat (ES-Tat-cNPs) were about 3-fold and 4-fold higher than those for the nanoparticles loading only insulin (ES-cNPs), respectively. The evaluations in vivo of ES-Tat-cNPs were conducted on diabetic rats and normal minipigs, respectively. The experimental results on rats revealed that the pharmacodynamical bioavailability of ES-Tat-cNPs had 2.16-fold increase compared with ES-cNPs. After oral administration of nanoparticle suspensions to the minipigs, insulin bioavailability of ES-Tat-cNPs was 1.73-fold higher than that of ES-cNPs, and the main absorption site of insulin was probably located in the colon for the two nanoparticles. In summary, this report provided an exploratory means for the improvement of oral absorption of insulin.

  10. Tumor acidity-activatable TAT targeted nanomedicine for enlarged fluorescence/magnetic resonance imaging-guided photodynamic therapy.

    PubMed

    Gao, Meng; Fan, Feng; Li, Dongdong; Yu, Yue; Mao, Kuirong; Sun, Tianmeng; Qian, Haisheng; Tao, Wei; Yang, Xianzhu

    2017-07-01

    Nanoparticles simultaneously integrated the photosensitizers and diagnostic agents represent an emerging approach for imaging-guided photodynamic therapy (PDT). However, the diagnostic sensitivity and therapeutic efficacy of nanoparticles as well as the heterogeneity of tumors pose tremendous challenges for clinical imaging-guided PDT treatment. Herein, a polymeric nanoparticle with tumor acidity (pH e )-activatable TAT targeting ligand that encapsulates the photosensitizer chlorin e6 (Ce6) and chelates contrast agent Gd 3+ is successfully developed for fluorescence/magnetic resonance (MR) dual-model imaging-guided precision PDT. We show clear evidence that the resulting nanoparticle DA TAT-NP [its TAT lysine residues' amines was modified by 2,3-dimethylmaleic anhydride (DA)] efficiently avoids the rapid clearance by reticuloendothelial system (RES) by masking of the TAT peptide, resulting in the significantly prolonged circulation time in the blood. Once accumulating in the tumor tissues, DA TAT-NP is reactivated by tumor acidity to promote cellular uptake, resulting in enlarged fluorescence/MR imaging signal intensity and elevated in vivo PDT therapeutic effect. This concept provides new avenues to design tumor acidity-activatable targeted nanoparticles for imaging-guided cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

    PubMed

    Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Fanales-Belasio, Emanuele; Moretti, Sonia; Sernicola, Leonardo; Cara, Andrea; Negri, Donatella R M; Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Scoglio, Arianna; Caputo, Antonella; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Ciccozzi, Massimo; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

    2004-09-03

    Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

  12. Improving ED specimen TAT using Lean Six Sigma.

    PubMed

    Sanders, Janet H; Karr, Tedd

    2015-01-01

    Lean and Six Sigma are continuous improvement methodologies that have garnered international fame for improving manufacturing and service processes. Increasingly these methodologies are demonstrating their power to also improve healthcare processes. The purpose of this paper is to discuss a case study for the application of Lean and Six Sigma tools in the reduction of turnaround time (TAT) for Emergency Department (ED) specimens. This application of the scientific methodologies uncovered opportunities to improve the entire ED to lab system for the specimens. This case study provides details on the completion of a Lean Six Sigma project in a 1,000 bed tertiary care teaching hospital. Six Sigma's Define, Measure, Analyze, Improve, and Control methodology is very similar to good medical practice: first, relevant information is obtained and assembled; second, a careful and thorough diagnosis is completed; third, a treatment is proposed and implemented; and fourth, checks are made to determine if the treatment was effective. Lean's primary goal is to do more with less work and waste. The Lean methodology was used to identify and eliminate waste through rapid implementation of change. The initial focus of this project was the reduction of turn-around-times for ED specimens. However, the results led to better processes for both the internal and external customers of this and other processes. The project results included: a 50 percent decrease in vials used for testing, a 50 percent decrease in unused or extra specimens, a 90 percent decrease in ED specimens without orders, a 30 percent decrease in complete blood count analysis (CBCA) Median TAT, a 50 percent decrease in CBCA TAT Variation, a 10 percent decrease in Troponin TAT Variation, a 18.2 percent decrease in URPN TAT Variation, and a 2-5 minute decrease in ED registered nurses rainbow draw time. This case study demonstrated how the quantitative power of Six Sigma and the speed of Lean worked in harmony to improve

  13. HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.

    PubMed

    Doublier, Sophie; Zennaro, Cristina; Spatola, Tiziana; Lupia, Enrico; Bottelli, Antonella; Deregibus, Maria Chiara; Carraro, Michele; Conaldi, Pier Giulio; Camussi, Giovanni

    2007-02-19

    To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

  14. Exosomal miR-9 Released from HIV Tat Stimulated Astrocytes Mediates Microglial Migration.

    PubMed

    Yang, Lu; Niu, Fang; Yao, Honghong; Liao, Ke; Chen, Xufeng; Kook, Yeonhee; Ma, Rong; Hu, Guoku; Buch, Shilpa

    2018-03-01

    Chronic neuroinflammation still remains a common underlying feature of HIV-infected patients on combined anti-retroviral therapy (cART). Previous studies have reported that despite near complete suppression of virus replication by cART, cytotoxic viral proteins such as HIV trans-activating regulatory protein (Tat) continue to persist in tissues such as the brain and the lymph nodes, thereby contributing, in part, to chronic glial activation observed in HIV-associated neurological disorders (HAND). Understanding how the glial cells cross talk to mediate neuropathology is thus of paramount importance. MicroRNAs (miR) also known as regulators of gene expression, have emerged as key paracrine signaling mediators that regulate disease pathogenesis and cellular crosstalk, through their transfer via the extracellular vesicles (EV). In the current study we have identified a novel function of miR-9, that of mediating microglial migration. We demonstrate that miR-9 released from Tat-stimulated astrocytes can be taken up by microglia resulting in their migratory phenotype. Exposure of human astrocytoma (A172) cells to HIV Tat resulted in induction and release of miR-9 in the EVs, which, was taken up by microglia, leading in turn, increased migration of the latter cells, a process that could be blocked by both an exosome inhibitor GW4869 or a specific target protector of miR-9. Furthermore, it was also demonstrated that EV miR-9 mediated inhibition of the expression of target PTEN, via its binding to the 3'UTR seed sequence of the PTEN mRNA, was critical for microglial migration. To validate the role of miR-9 in this process, microglial cells were treated with EVs loaded with miR-9, which resulted in significant downregulation of PTEN expression with a concomitant increase in microglial migration. These findings were corroborated by transfecting microglia with a specific target protector of PTEN, that blocked miR-9-mediated downregulation of PTEN as well as microglial

  15. Identification of amino acids that promote specific and rigid TAR RNA-tat protein complex formation.

    PubMed

    Edwards, Thomas E; Robinson, Bruce H; Sigurdsson, Snorri Th

    2005-03-01

    The Tat protein and the transactivation responsive (TAR) RNA form an essential complex in the HIV lifecycle, and mutations in the basic region of the Tat protein alter this RNA-protein molecular recognition. Here, EPR spectroscopy was used to identify amino acids, flanking an essential arginine of the Tat protein, which contribute to specific and rigid TAR-Tat complex formation by monitoring changes in the mobility of nitroxide spin-labeled TAR RNA nucleotides upon binding. Arginine to lysine N-terminal mutations did not affect TAR RNA interfacial dynamics. In contrast, C-terminal point mutations, R56 in particular, affected the mobility of nucleotides U23 and U38, which are involved in a base-triple interaction in the complex. This report highlights the role of dynamics in specific molecular complex formation and demonstrates the ability of EPR spectroscopy to study interfacial dynamics of macromolecular complexes.

  16. The HIV-1 associated protein, Tat1–86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: a no-net-flux microdialysis study

    PubMed Central

    Ferris, Mark J.; Frederick-Duus, Danielle; Fadel, Jim; Mactutus, Charles F.; Booze, Rosemarie M.

    2009-01-01

    Injection drug use accounts for approximately one-third of HIV-infections in the United States. HIV associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 hours of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 hrs post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharamacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to DA systems occurring in NeuroAIDS. PMID:19344635

  17. The Taming of the Cell Penetrating Domain of the HIV Tat: Myths and Realities

    PubMed Central

    Chauhan, Ashok; Tikoo, Akshay; Kapur, Arvinder K.; Singh, Mahavir

    2007-01-01

    Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV-Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. Inspite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences. PMID:17196289

  18. PPAR agonist-mediated protection against HIV Tat-induced cerebrovascular toxicity is enhanced in MMP-9-deficient mice

    PubMed Central

    Huang, Wen; Chen, Lei; Zhang, Bei; Park, Minseon; Toborek, Michal

    2014-01-01

    The strategies to protect against the disrupted blood–brain barrier (BBB) in HIV-1 infection are not well developed. Therefore, we investigated the potential of peroxisome proliferator-activated receptor (PPAR) agonists to prevent enhanced BBB permeability induced by HIV-1-specific protein Tat. Exposure to Tat via the internal carotid artery (ICA) disrupted permeability across the BBB; however, this effect was attenuated in mice treated with fenofibrate (PPARα agonist) or rosiglitazone (PPARγ agonist). In contrast, exposure to GW9662 (PPARγ antagonist) exacerbated Tat-induced disruption of the BBB integrity. Increased BBB permeability was associated with decreased tight junction (TJ) protein expression and activation of ERK1/2 and Akt in brain microvessels; these effects were attenuated by cotreatment with fenofibrate but not with rosiglitazone. Importantly, both PPAR agonists also protected against Tat-induced astrogliosis and neuronal loss. Because disruption of TJ integrity has been linked to matrix metalloproteinase (MMP) activity, we also evaluated Tat-induced effects in MMP-9-deficient mice. Tat-induced cerebrovascular toxicity, astrogliosis, and neuronal loss were less pronounced in MMP-9-deficient mice as compared with wild-type controls and were further attenuated by PPAR agonists. These results indicate that enhancing PPAR activity combined with targeting MMPs may provide effective therapeutic strategies in brain infection by HIV-1. PMID:24424383

  19. Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

    PubMed

    Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

    2001-12-13

    We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential

  20. Allosteric regulation of rhomboid intramembrane proteolysis.

    PubMed

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-09-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

  1. Allosteric regulation of rhomboid intramembrane proteolysis

    PubMed Central

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-01-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

  2. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    PubMed

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-06-30

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

  3. Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

    PubMed Central

    Park, Jung-Sun; Park, Soo-Young; Cho, Hyun-Il; Sohn, Hyun-Jung

    2011-01-01

    Background Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining. Results DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines. PMID:21860612

  4. Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

    PubMed Central

    Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2014-01-01

    Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation. PMID

  5. Is the Achievement Motive Gender-Biased? The Validity of TAT/PSE in Women and Men

    PubMed Central

    Gruber, Nicole

    2017-01-01

    In picture story exercises like the Thematic Apperception Test (TAT; Heckhausen, 1963), different pictures are presented to a person with the instruction to create a story using the scenes portrayed in the image. It is assumed, that people identify themselves with the people in the images and project their unconscious motives (e.g., achievement motive) onto them. As the TAT shows only men in the pictures, critics claimed the test is gender-biased; assuming women cannot identify with men in pictures. However, it was not assessed, whether female protagonists of the picture really trigger the same achievement motive as men. Therefore, two studies were conducted to address the gender difference and validity of the TAT using a version with only men in the pictures (study 1) or only women in the pictures (study 2). The results shows that the original TAT of Heckhausen is a valid instrument for women and men, but the modified version with only women in the pictures cannot validly measure the achievement motive in the male sample. PMID:28261126

  6. Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

    PubMed

    Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

    2016-07-12

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  7. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

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

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrolmore » induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.« less

  8. The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters.

    PubMed

    Rezza, Giovanni; Fiorelli, Valeria; Dorrucci, Maria; Ciccozzi, Massimo; Tripiciano, Antonella; Scoglio, Arianna; Collacchi, Barbara; Ruiz-Alvarez, Maria; Giannetto, Concettina; Caputo, Antonella; Tomasoni, Lina; Castelli, Francesco; Sciandra, Mauro; Sinicco, Alessandro; Ensoli, Fabrizio; Buttò, Stefano; Ensoli, Barbara

    2005-04-15

    The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.

  9. Benefit-Cost Analysis of TAT Phase I Worker Training. Training and Technology Project. Special Report.

    ERIC Educational Resources Information Center

    Kirby, Frederick C.; Castagna, Paul A.

    The purpose of this study is to estimate costs and benefits and to compute alternative benefit-cost ratios for both the individuals and the Federal Government as a result of investing time and resources in the Training and Technology (TAT) Project. TAT is a continuing experimental program in training skilled workers for private industry. The five…

  10. HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

    PubMed

    El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

    2018-05-01

    Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

    PubMed

    Tan, Elaine Y M; Law, Janice W S; Wang, Chi-Hwa; Lee, Alan Y W

    2007-12-01

    Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth

  12. Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

    PubMed

    Vilches, Clara; Boiadjieva-Knöpfel, Emilia; Bodoy, Susanna; Camargo, Simone; López de Heredia, Miguel; Prat, Esther; Ormazabal, Aida; Artuch, Rafael; Zorzano, Antonio; Verrey, François; Nunes, Virginia; Palacín, Manuel

    2018-04-02

    Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y + LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo , we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y + LAT1/CD98hc. Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo , and y + LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans. Copyright © 2018 by the American Society of Nephrology.

  13. Utilization of Bacillus sp. strain TAT105 as a biological additive to reduce ammonia emissions during composting of swine feces.

    PubMed

    Kuroda, Kazutaka; Waki, Miyoko; Yasuda, Tomoko; Fukumoto, Yasuyuki; Tanaka, Akihiro; Nakasaki, Kiyohiko

    2015-01-01

    Bacillus sp. strain TAT105 is a thermophilic, ammonium-tolerant bacterium that grows assimilating ammonium nitrogen and reduces ammonia emission during composting of swine feces. To develop a practical use of TAT105, a dried solid culture of TAT105 (5.3 × 10(9) CFU/g of dry matter) was prepared as an additive. It could be stored for one year without significant reduction of TAT105. Laboratory-scale composting of swine feces was conducted by mixing the additive. When the additive, mixed with an equal weight of water one day before use, was added to obtain a TAT105 concentration of above 10(7) CFU/g of dry matter in the initial material, the ammonia concentration emitted was lower and nitrogen loss was approximately 22% lower in the treatment with the additive than in the control treatment without the additive. The colony formation on an agar medium containing high ammonium could be used for enumeration of TAT105 in the composted materials.

  14. Neonatal hippocampal Tat injections: developmental effects on prepulse inhibition (PPI) of the auditory startle response

    PubMed Central

    Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

    2013-01-01

    The current estimate of children (<15 years) living with HIV and AIDS is 2.2 million [UNAIDS/WHO, 2005. AIDS Epidemic Update. UNAIDS, Geneva]. The major source of infection occurs through vertical transmission of the virus from mother to child during delivery [UNAIDS/ WHO, 2005. AIDS Epidemic Update. UNAIDS, Geneva]. Recent studies have shown that timing of HIV-1 infection might be related to the onset and rate of progression of CNS disease [Blanche, S., Mayaux, M.-J., Rouziox, C., Teglas, J.-P., Firtion, G., Monpoux, F., Cicaru-Vigneron, N., Meier, F., Tricoire, J., Courpotin, C., Vilmer, E., Griscelli, C., Delfraissy, J.-F., 1994. Relation of the course of HIV infection in children to the severity of the disease in their mothers at delivery. N. Engl. J. Med. 330 (5), 308–312]. The effects of HIV on the brain are thought to be mediated indirectly through the viral toxins Tat and gp120. This study characterized developmental effects on PPI following intrahippocampal administration of Tat. On postnatal day (P)1, one male and one female pup from each of eight Sprague–Dawley litters were bilaterally injected with 50 µg Tat or saline (1 µl volume). Animals were tested for PPI of the auditory startle response (ASR) (ISIs of 0, 8,40, 80, 120, and 4000 ms, six trial blocks, Latin-square design) on days 30, 60 and 90. Tat altered PPI and the pattern of alterations was different for males and females. For males, a leftward shift was evident in the ISI for maximal inhibition of the response on day 30 and on day 60 (χ2(1) = 4.7,p ≤ .03, and χ2(1) = 5.3, p ≤ .02, respectively), but not on day 90. For females, Tat altered peak ASR latency across PPI trials (8–120 ms) at all days of testing (30, 60, and 90 days of age), as indexed by orthogonal component analyses, indicating less modulation of PPI by ISI. Data collected from a second group that were tested only once at 90 days of age, suggested that the observed adverse Tat effects for males and females early in

  15. Protective effects of intraperitoneal injection of TAT-SOD against focal cerebral ischemia/reperfusion injury in rats.

    PubMed

    Ye, Nanhui; Liu, Shutao; Lin, Yanyun; Rao, Pingfan

    2011-12-05

    The intracellular superoxide anion has been shown to be involved in brain injury. TAT-Superoxide dismutase (TAT-SOD) can be transduced across the cell membrane to scavenge superoxide. This protein's unique properties make it a promising therapeutic candidate to attenuate cerebral damage. In this study, we sought further the understanding of the fusion protein's cerebral protective effects and the mechanism which is exerted in these effects. Male Sprague Dawley rats (n=100, 230±20 g) were divided randomly into five experimental groups: a sham group, a cerebral Ischemia/Reperfusion (I/R) group treated with saline (20 ml/Kg, i.p.), and three cerebral I/R groups treated with TAT-SOD (25 KU/ml/Kg, i.p.) at either 2h before I/R, 2h after I/R or 4h after I/R. Cerebral I/R injury was facilitated by inducing ischemia for two hours followed by 24h reperfusion. The levels of SOD, Malondialdehyde (MDA), and ATPase in cerebral tissues were determined. The apoptotic indexes were evaluated, and apoptosis genes were analyzed immunohistochemically. TAT-SOD treatment significantly increased cerebral SOD and ATPase activities, decreased MDA content, and remarkably reduced apoptosis indexes. TAT-SOD treatments 2h before or after I/R significantly reduced caspase-3 and bax proteins and boosted bcl-2 protein, while the treatment at 4h after I/R showed no influence on the three proteins. TAT-SOD treatment effectively enhanced cerebral antioxidant ability, reduced lipid peroxidation, preserved mitochondrial ATPase and thus inhibited nerve cell apoptosis. The effective treatment window extended from 2h before to 2h after I/R. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Characterization of free radical defense system in high glucose cultured HeLa-tat cells: consequences for glucose-induced cytotoxicity.

    PubMed

    Bouvard, Sophie; Faure, Patrice; Roucard, Corinne; Favier, Alain; Halimi, Serge

    2002-09-01

    HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.

  17. The anti-cancer drug Sunitinib promotes autophagy and protects from neurotoxicity in an HIV-1 Tat model of neurodegeneration

    PubMed Central

    Fields, Jerel A.; Metcalf, Jeff; Overk, Cassia; Adame, Anthony; Spencer, Brian; Wrasidlo, Wolfgang; Florio, Jazmin; Rockenstein, Edward; He, Johnny J.; Masliah, Eliezer

    2017-01-01

    Despite the success of antiretroviral therapies to control systemic HIV-1 infection, the prevalence of HIV-associated neurocognitive disorders (HAND) has not decreased among aging patients with HIV. Autophagy pathway alterations, triggered by HIV-1 proteins including gp120, Tat, and Nef, might contribute to the neurodegenerative process in aging patients with HAND. Although no treatments are currently available to manage HAND, we have previously shown that Sunitinib, an anti-cancer drug that blocks receptor tyrosine-kinase and cyclin kinase pathways, might be of interest. Studies in cancer models suggest that sunitinib might also modulate autophagy, which is dysregulated in our models of Tat-induced neurotoxicity. We evaluated the efficacy of sunitinib to promote autophagy in the CNS and ameliorate neurodegeneration using LC3-GFP expressing neuronal cells challenged with low concentrations of Tat and using inducible Tat transgenic mice. In neuronal cultures challenged with low levels of Tat, sunitinib increased markers of autophagy such as LC3-II and reduced p62 accumulation in a dose-dependent manner. In vivo, sunitinib treatment restored LC3-II, p62, and Endophilin B1 (EndoB1) levels in doxycycline-induced Tat transgenic mice. Moreover, in these animals sunitinib reduced the hyperactivation of CDK5, tau hyper-phosphorylation and p35 cleavage to p25. Restoration of CDK5 and autophagy were associated with reduced neurodegeneration and behavioral alterations. Alterations in autophagy in the Tat tg mice were associated with reduced levels of a CDK5 substrate, EndoB1, and levels of total EndoB1 were normalized by sunitinib treatment. We conclude that sunitinib might ameliorate Tat-mediated autophagy alterations and may decrease neurodegeneration in aging patients with HAND. PMID:28105557

  18. Sequential delivery of TAT-HSP27 and VEGF using microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

    PubMed

    Shin, Seung-Hwa; Lee, Jangwook; Lim, Kwang Suk; Rhim, Taiyoun; Lee, Sang Kyung; Kim, Yong-Hee; Lee, Kuen Yong

    2013-02-28

    Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats

    PubMed Central

    Brailoiu, Eugen; Deliu, Elena; Sporici, Romeo A.; Benamar, Khalid

    2014-01-01

    The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1–86) produced a concentration-dependent increase in cytosolic Ca2+ in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca2+ elevation: 1) lysosomal Ca2+ mobilization, 2) Ca2+ release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca2+ influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca2+ entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy. PMID:24694382

  20. Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS.

    PubMed

    Paris, Jason J; Fenwick, Jason; McLaughlin, Jay P

    2014-05-01

    Increased anxiety is co-morbid with human immunodeficiency virus (HIV) infection. Actions of the neurotoxic HIV-1 regulatory protein, Tat, may contribute to affective dysfunction. We hypothesized that Tat expression would increase anxiety-like behavior of female GT-tg bigenic mice that express HIV-1 Tat protein in the brain in a doxycycline-dependent manner. Furthermore, given reports that HIV-induced anxiety may occur at lower rates among women, and that the neurotoxic effects of Tat are ameliorated by sex steroids in vitro, we hypothesized that 17β-estradiol and/or progesterone would ameliorate Tat-induced anxiety-like effects. Among naturally-cycling proestrous and diestrous mice, Tat-induction via 7days of doxycycline treatment significantly increased anxiety-like responding in an open field, elevated plus maze and a marble-burying task, compared to treatment with saline. Proestrous mice demonstrated less anxiety-like behavior than diestrous mice in the open field and elevated plus maze, but these effects did not significantly interact with Tat-induction. Among ovariectomized mice, doxycycline-induced Tat protein significantly increased anxiety-like behavior in an elevated plus maze and a marble burying task compared to saline-treated mice, but not an open field (where anxiety-like responding was already maximal). Co-administration of progesterone (4mg/kg), but not 17β-estradiol (0.09mg/kg), with doxycycline significantly ameliorated anxiety-like responding in the elevated plus maze and marble burying tasks. When administered together, 17β-estradiol partially antagonized the protective effects of progesterone on Tat-induced anxiety-like behavior. These findings support evidence of steroid-protection over HIV-1 proteins, and extend them by demonstrating the protective capacity of progesterone on Tat-induced anxiety-like behavior of ovariectomized female mice. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. A mini-review of TAT-MyoD fused proteins: state of the art and problems to solve.

    PubMed

    Patruno, Marco; Melotti, Luca; Gomiero, Chiara; Sacchetto, Roberta; Topel, Ohad; Martinello, Tiziana

    2017-12-05

    The transcriptional activator TAT is a small peptide essential for viral replication and possesses the property of entering the cells from the extracellular milieu, acting as a membrane shuttle. In order to safely differentiate cells an innovative methodology, based on the fusion of transcription factors and the TAT sequence, is discussed in this short review. In several studies, it has been demonstrated that TAT protein can be observed in the cell nucleus after few hours from the inoculation although its way of action is not fully understood. However, further studies will be necessary to develop this methodology for clinical purposes.

  2. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli.

    PubMed

    Caldinelli, Laura; Albani, Diego; Pollegioni, Loredano

    2013-04-04

    Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.

  3. SjTat-TPI facilitates adaptive T-cell responses and reduces hepatic pathology during Schistosoma japonicum infection in BALB/c mice.

    PubMed

    Zhang, Wenyue; Luo, Xiaofeng; Zhang, Fan; Zhu, Yuxiao; Yang, Bingya; Hou, Min; Xu, Zhipeng; Yu, Chuanxin; Chen, Yingying; Chen, Lin; Ji, Minjun

    2015-12-30

    Schistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. China is one of the countries most affected by Schistosoma japonicum and an effective vaccine is still needed. In this study, we adopted Tat-mediated protein transduction technology to investigate the impact of different antigen presented approaches on host's immune response and the potential protection against Schistosoma japonicum infection. We successfully constructed the recombinant S. japonicum triosephosphate isomerase, Tat-TPI, as a vaccine candidate. Whether injected with Tat-TPI in foot pad or vaccinated with Tat-TPI in the back subcutaneously for three times, the draining popliteal lymph nodes and spleen both developed a stronger CD8(+)T response (Tc1) in mice. Not only that, but it also helped CD4(+)T cells to produce more IFN-γ than TPI immunisation. In addition, it could boost IgG production, especially IgG1 subclass. Most importantly, Tat-TPI immunisation led to the significant smaller area of a single egg granuloma in the livers as compared with TPI-vaccinated or control groups. However, the anti-infection efficiency induced by Tat-TPI was still restricted. This study indicated that immunisation with Tat-fused TPI could contribute to enhance CD4(+)T-cell response and decrease hepatic egg granulomatous area after S. japonicum infection though it did not achieve our expected protection against Schistosoma japonicum infection. The optimal vaccine strategy warrants further research.

  4. Passage of Magnetic Tat-Conjugated Fe3O4@SiO2 Nanoparticles Across In Vitro Blood-Brain Barrier

    NASA Astrophysics Data System (ADS)

    Zhao, Xueqin; Shang, Ting; Zhang, Xiaodan; Ye, Ting; Wang, Dajin; Rei, Lei

    2016-10-01

    Delivery of diagnostic or therapeutic agents across the blood-brain barrier (BBB) remains a major challenge of brain disease treatment. Magnetic nanoparticles are actively being developed as drug carriers due to magnetic targeting and subsequently reduced off-target effects. In this paper, we developed a magnetic SiO2@Fe3O4 nanoparticle-based carrier bound to cell-penetrating peptide Tat (SiO2@Fe3O4 -Tat) and studied its fates in accessing BBB. SiO2@Fe3O4-Tat nanoparticles (NPs) exhibited suitable magnetism and good biocompatibility. NPs adding to the apical chamber of in vitro BBB model were found in the U251 glioma cells co-cultured at the bottom of the Transwell, indicating that particles passed through the barrier and taken up by glioma cells. Moreover, the synergistic effects of Tat and magnetic field could promote the efficient cellular internalization and the permeability across the barrier. Besides, functionalization with Tat peptide allowed particles to locate into the nucleus of U251 cells than the non-conjugated NPs. These results suggest that SiO2@Fe3O4-Tat NPs could penetrate the BBB through the transcytosis of brain endothelial cells and magnetically mediated dragging. Therefore, SiO2@Fe3O4-Tat NPs could be exploited as a potential drug delivery system for chemotherapy and gene therapy of brain disease.

  5. Sequence conservation and antibody cross-recognition of clade B human immunodeficiency virus (HIV) type 1 Tat protein in HIV-1-infected Italians, Ugandans, and South Africans.

    PubMed

    Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Ruiz-Alvarez, Maria J; Scoglio, Arianna; Ensoli, Fabrizio; Ciccozzi, Massimo; Collacchi, Barbara; Sabbatucci, Michela; Cafaro, Aurelio; Guzmán, Carlos A; Borsetti, Alessandra; Caputo, Antonella; Vardas, Eftyhia; Colvin, Mark; Lukwiya, Matthew; Rezza, Giovanni; Ensoli, Barbara

    2003-10-15

    We determined immune cross-recognition and the degree of Tat conservation in patients infected by local human immunodeficiency virus (HIV) type 1 strains. The data indicated a similar prevalence of total and epitope-specific anti-Tat IgG in 578 serum samples from HIV-infected Italian (n=302), Ugandan (n=139), and South African (n=137) subjects, using the same B clade Tat protein that is being used in vaccine trials. In particular, anti-Tat antibodies were detected in 13.2%, 10.8%, and 13.9% of HIV-1-infected individuals from Italy, Uganda, and South Africa, respectively. Sequence analysis results indicated a high similarity of Tat from the different circulating viruses with BH-10 Tat, particularly in the 1-58 amino acid region, which contains most of the immunogenic epitopes. These data indicate an effective cross-recognition of a B-clade laboratory strain-derived Tat protein vaccine by individuals infected with different local viruses, owing to the high similarity of Tat epitopes.

  6. Tat-CBR1 inhibits inflammatory responses through the suppressions of NF-κB and MAPK activation in macrophages and TPA-induced ear edema in mice

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

    Kim, Young Nam; Kim, Dae Won; Jo, Hyo Sang

    Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB)more » and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases. - Highlights: • Transduced Tat-CBR1 reduces LPS-induced inflammatory mediators and cytokines. • Tat-CBR1 inhibits MAPK and NF-κB activation. • Tat-CBR1 ameliorates inflammation response in vitro and in vivo. • Tat-CBR1 may be useful as potential therapeutic agent for inflammation.« less

  7. Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages.

    PubMed Central

    Murphy, K M; Sweet, M J; Ross, I L; Hume, D A

    1993-01-01

    The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells

  8. Detection of human immunodeficiency virus type 1 (HIV-1) Tat protein by aptamer-based biosensors

    NASA Astrophysics Data System (ADS)

    Hashim, Uda; Fatin, M. F.; Ruslinda, A. R.; Gopinath, Subash C. B.; Uda, M. N. A.

    2017-03-01

    A study was conducted to detect the human immunodeficiency virus (HIV-1) Tat protein using interdigitated electrodes. The measurements and images of the IDEs' finger gaps and the images of chitosan-carbon nanotubes deposited on top of the interdigitated electrodes were taken using the Scanning Electron Microscope. The detection of HIV-1 Tat protein was done using split aptamers and aptamer tail. Biosensors were chosen as diagnostic equipment due to their rapid diagnostic capabilities.

  9. HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines.

    PubMed

    El-Hage, Nazira; Wu, Guanghan; Wang, Juan; Ambati, Jayakrishna; Knapp, Pamela E; Reed, Janelle L; Bruce-Keller, Annadora J; Hauser, Kurt F

    2006-01-15

    Opiates exacerbate human immunodeficiency virus type 1 (HIV-1) Tat(1-72)-induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, the role of MCP-1 in neuro-acquired immunodeficiency syndrome (neuroAIDS) progression in opiate abusers, or nonabusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was found to be significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat(1-72) than in vehicle-, mu-opioid receptor (MOR) antagonist-, or inactive, mutant Tat(delta31-61)-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1(-/-) astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen after treatment with Tat alone, or with morphine plus inactive Tat(delta31-61) or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity after morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and

  10. Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

    PubMed Central

    Chin, D J; Selby, M J; Peterlin, B M

    1991-01-01

    Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription. Images PMID:1900539

  11. TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors

    PubMed Central

    Sethuraman, Vijay A; Bae, You Han

    2007-01-01

    A novel drug targeting system for acidic solid tumors has been developed based on ultra pH sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of Poly(L-lactic acid) (PLLA) and a hydrophilic shell consisting of Polyethylene Glycol (PEG) conjugated to TAT (TATmicelle), 2) An ultra pH sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TATmicelles had particle sizes between 20 to 45 nm and their critical micelle concentrations were 3.5 mg/L to 5.5 mg/L. The TATmicelles, upon mixing with pH sensitive PSD-b-PEG, showed slight increase in particle size between pH 8.0 and 6.8 (60–90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flowcytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The flowcytometry indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above drug loaded micelles would be able to target any hydrophobic drug near the nucleus. PMID:17239466

  12. The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Kurosaka, Goyu; Abe, Fumiyoshi

    2018-01-01

    In the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25 MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10-25 MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25 MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25 MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10-25 MPa.

  13. The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

    PubMed Central

    Benjak, Andrej; Uplekar, Swapna; Rougemont, Jacques; Guilhot, Christophe; Malaga, Wladimir; Martín, Carlos; Cole, Stewart T.

    2014-01-01

    The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. PMID:24874799

  14. Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes.

    PubMed

    Soto, Ana Maria; Marky, Luis A

    2002-10-15

    Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions.

  15. [Keap1-tat peptide attenuates oxidative stress damage in hippocampal CA1 region and learning and memory deficits following global cerebral ischemia].

    PubMed

    Tu, Jing-yi; Zhu, Ying; Shang, Shu-ling; Zhang, Xi; Tang, Hui; Wang, Rui-min

    2016-02-18

    To design Keap1-tat peptide and explore its neuroprotective role on hipocampal CA1 neuron, as well as the effect on spacial learning and memory function following global cerebral ischemia. Adult male Sprague Dawley (SD) rats were subjected to global cerebral ischemia (GCI) by four-vessel occlusion for 15 min and randomly divided into five groups: sham, sham+Keap1-tat, ischemia/reperfusion (I/R), Keap1-tat peptide- and vehicle-administrated groups. For Keap1-tat or vehicle groups, the rats were treated with Keap1-tat (30, 50, 100 μg in 5 μL 0.9% saline) or the same volume vehicle by intracerebroventricular injection (icv) 30 min prior to ischemia. Cresyl violet staining was used to observe the surviving neurons and 4-hydroxy-2-noneal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunostaining were used to detect the change of markers response to oxidative stress in hippocampal CA1 region. The spatial learning and memory function of the rats was evaluated using Morris water maze. Compared with sham group, the number of surviving neurons in ischemia-reperfusion and vehicle groups significantly decreased in the hippocampal CA1 region (P<0.05), while administration of Keap1-tat significantly decreased the damage following GCI (P<0.05), and the dose of 50 μg existed the most effective neuroprotective role. Furthermore, immunostaining intensity of 4-HNE and 8-OHdG, markers of oxidative stress damage attenuated by Keap1-tat peptide as compared with vehicle group in CA1 region. Of significant interest, the time of finding underwater platform in Keap1-tat group animals was significantly short, and after removing the platform, the probe time of Keap1-tat group animals in the original quadrant where the platform was significantly increased compared with that of vehicle and I/R group animals (P<0.05). Keap1-tat peptide can effectively attenuate neuronal damage in hippocampal CA1 region and improve learning and memory function, which might bedue to the attenuation of

  16. Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction.

    PubMed

    Kim, Hye Jung; Kim, Myoung-Hun; Kim, Jong Tae; Lee, Won-Jin; Kim, Eunjung; Lim, Kwang Suk; Kim, Jang Kyoung; Yang, Young Il; Park, Ki Dong; Kim, Yong-Hee

    2015-10-10

    Myocardial infarction (MI) results in the substantial loss of functional cardiomyocytes, which frequently leads to intractable heart disorders. Cardiac stem cells (CSCs) that retain the capacity to replace all cardiac cells might be a promising strategy for providing a source of new functional cardiomyocytes; however, the poor survival and engraftment of transplanted CSCs in the hostile environment of MI critically mitigate their therapeutic benefits. To capitalize their therapeutic potential, an ex vivo strategy in which CSCs were introduced to the recombinant heat shock protein 27 (Hsp27) through a TAT protein transduction domain for increasing the viability and engraftment in the infarcted myocardium was designed. A recombinant TAT fused Hsp27 (TAT-Hsp27) was able to enter CSCs in a dose-dependent manner. CSCs transduced with TAT-Hsp27 expressed not only endogenous Hsp27 but externally introduced Hsp27, resulting in substantial increase of their anti-oxidative and anti-apoptotic properties via suppressing reactive oxygen species production, the MAPKs signaling pathway, and caspase activation. TAT-Hsp27 enabled CSCs to be protected from apoptotic- and hypoxic-induced cell death during in vitro cardiomyogenic differentiation. In vivo studies demonstrated that CSCs transduced TAT-Hsp27 significantly increased the survival and engraftment in the acutely infarcted myocardium, which is closely related to caspase activity suppression. Finally, CSCs transduced TAT-Hsp27 improved cardiac function and attenuated cardiac remodeling in comparison with non-transduced CSCs. Overall, our approach, which is based on the ex vivo intracellular transduction of TAT-Hsp27 into CSCs before myocardial delivery, might be effective in treating MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Negotiation of intracellular membrane barriers by TAT-modified gold nanoparticles.

    PubMed

    Krpetić, Zeljka; Saleemi, Samia; Prior, Ian A; Sée, Violaine; Qureshi, Rumana; Brust, Mathias

    2011-06-28

    This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.

  18. HIV Tat1-72 and Opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines

    PubMed Central

    El-Hage, Nazira; Wu, Guanghan; Wang, Juan; Ambati, Jayakrishna; Knapp, Pamela E.; Reed, Janelle L.; Bruce-Keller, Annadora J.; Hauser, Kurt F.

    2011-01-01

    Opiates exacerbate HIV-1 Tat1-72–induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein-1 (MCP-1 or CCL2), in particular, is potentiated by opiate-HIV Tat interactions in vitro. Although MCP-1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, its role in neuroAIDS progression in opiate abusers, or non-abusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat1-72 than in vehicle-, μ opioid receptor (MOR) antagonist-, or inactive, mutant TatΔ31-61-treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP-1 antibodies, or medium from MCP-1−/− astrocytes. In the presence of morphine (time-release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post-injection. This was not seen following treatment with Tat alone, or with morphine plus inactive TatΔ31-61 or naltrexone. Glia displayed increased MOR and MCP-1 immunoreactivity following morphine and/or Tat exposure. The findings indicate that MCP-1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial-derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate-driven amplification/positive feedback of MCP-1 production and inflammation. PMID:16206161

  19. HIV-Tat immunization induces cross-clade neutralizing antibodies and CD4(+) T cell increases in antiretroviral-treated South African volunteers: a randomized phase II clinical trial.

    PubMed

    Ensoli, Barbara; Nchabeleng, Maphoshane; Ensoli, Fabrizio; Tripiciano, Antonella; Bellino, Stefania; Picconi, Orietta; Sgadari, Cecilia; Longo, Olimpia; Tavoschi, Lara; Joffe, Daniel; Cafaro, Aurelio; Francavilla, Vittorio; Moretti, Sonia; Pavone Cossut, Maria Rosaria; Collacchi, Barbara; Arancio, Angela; Paniccia, Giovanni; Casabianca, Anna; Magnani, Mauro; Buttò, Stefano; Levendal, Elise; Ndimande, John Velaphi; Asia, Bennett; Pillay, Yogan; Garaci, Enrico; Monini, Paolo

    2016-06-09

    Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 μg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4(+) T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4(+) T-cell counts and therapy compliance. Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat

  20. Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification

    PubMed Central

    Garg, Himanshu; Joshi, Anjali

    2016-01-01

    Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

  1. Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

    PubMed

    Garg, Himanshu; Joshi, Anjali

    2016-05-01

    Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.

  2. A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

    PubMed Central

    Fujinaga, Koh; Irwin, Dan; Taube, Ran; Zhang, Fan; Geyer, Matthias; Peterlin, B. Matija

    2002-01-01

    The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex. PMID:12438619

  3. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    NASA Astrophysics Data System (ADS)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  4. Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

    PubMed Central

    Jayasena, S D; Johnston, B H

    1992-01-01

    tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

  5. Field Trial Data Analysis and Testing (FiTAT) Tool

    DTIC Science & Technology

    2011-10-01

    amplitude/phase pour chaque antenne du réseau) contenu dans les données acquises pourrait être faite par FiTAT et utilisé dans PAASoM pour déterminer...identity matrix, k is the loop gain, φ is the correlation matrix of the incident signals and T = [1 0 . . . 0]T . The length of vector T is equal to the

  6. Tat peptide and hexadecylphosphocholine introduction into pegylated liposomal doxorubicin: An in vitro and in vivo study on drug cellular delivery, release, biodistribution and antitumor activity.

    PubMed

    Teymouri, Manouchehr; Badiee, Ali; Golmohammadzadeh, Shiva; Sadri, Kayvan; Akhtari, Javad; Mellat, Mostafa; Nikpoor, Amin Reza; Jaafari, Mahmoud Reza

    2016-09-10

    We have investigated the co-addition of hexadecylphosphocholine (HePC) and a Tat derived peptide (Tat), coupled to Maleimide-PEG2000-DSPE pegylated liposomal doxorubicin (PLD) in many respects, including drug and liposome cellular delivery, drug release, biodistribution, in vivo cell delivery and antitumor activity. The liposomes were HePC-free and -containing liposomes, from which liposomes with 25, 50, 100 and 200 numbers of Tat/liposome were prepared. Similarly, DiI-C18 (3)-model liposomes (DiI-L and DiI-HePC-L) were prepared. HePC and Tat increased cellular delivery of Dox and cytotoxicity in B16F0 melanoma and C26 colon carcinoma cells. Tat enhanced liposome-cell interaction and caused Dox burst release. HePC and Tat reduced the serum retention time of liposomal Dox, slightly and dramatically, respectively. In comparison, Tat-liposomes enhanced Dox delivery to liver and spleen cells 3h post-injection. Likewise, Dox content of these tissues and tumor was lower at 24h. The naïve liposomes retarded tumor growth more effectively and their related median survival time of the treated C26 bearing BALB/c mice was longer than those of Tat-liposomes (MST>45days versus MST<38days). Overall liposomes exhibiting sustained drug release and negligible cell interaction were more suitable delivery systems in targeting cancerous tumors and suppressing their growth. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

    PubMed

    Liu, Dong-cai; Yang, Zhu-lin

    2011-11-01

    Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

    PubMed

    Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

  9. Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

    PubMed Central

    Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

  10. The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

    PubMed

    Moscatiello, Roberto; Sello, Simone; Novero, Mara; Negro, Alessandro; Bonfante, Paola; Navazio, Lorella

    2014-08-01

    Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  11. Regulation of selective autophagy: the p62/SQSTM1 paradigm.

    PubMed

    Lamark, Trond; Svenning, Steingrim; Johansen, Terje

    2017-12-12

    In selective autophagy, cytoplasmic components are selected and tagged before being sequestered into an autophagosome by means of selective autophagy receptors such as p62/SQSTM1. In this review, we discuss how selective autophagy is regulated. An important level of regulation is the selection of proteins or organelles for degradation. Components selected for degradation are tagged, often with ubiquitin, to facilitate recognition by autophagy receptors. Another level of regulation is represented by the autophagy receptors themselves. For p62, its ability to co-aggregate with ubiquitinated substrates is strongly induced by post-translational modifications (PTMs). The transcription of p62 is also markedly increased during conditions in which selective autophagy substrates accumulate. For other autophagy receptors, the LC3-interacting region (LIR) motif is regulated by PTMs, inhibiting or stimulating the interaction with ATG8 family proteins. ATG8 proteins are also regulated by PTMs. Regulation of the capacity of the core autophagy machinery also affects selective autophagy. Importantly, autophagy receptors can induce local recruitment and activation of ULK1/2 and PI3KC3 complexes at the site of cargo sequestration. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  12. Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae.

    PubMed

    Uemura, Satoshi; Mochizuki, Takahiro; Kurosaka, Goyu; Hashimoto, Takanori; Masukawa, Yuki; Abe, Fumiyoshi

    2017-10-01

    Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: Potential role of the thiol antioxidant N-acetylcysteine amide

    PubMed Central

    Banerjee, Atrayee; Zhang, Xinsheng; Manda, Kalyan Reddy; Banks, William A; Ercal, Nuran

    2010-01-01

    An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120+Tat+Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120+Tat+METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD. PMID:20188164

  14. Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR

    PubMed Central

    Sedore, Stanley C.; Byers, Sarah A.; Biglione, Sebastian; Price, Jason P.; Maury, Wendy J.; Price, David H.

    2007-01-01

    Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR. PMID:17576689

  15. Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

    PubMed Central

    Sumner-Smith, M; Roy, S; Barnett, R; Reid, L S; Kuperman, R; Delling, U; Sonenberg, N

    1991-01-01

    The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat. Images PMID:1895380

  16. Efficient induction of anti-tumor immunity by a TAT-CEA fusion protein vaccine with poly(I:C) in a murine colorectal tumor model.

    PubMed

    Park, Jung-Sun; Kim, Hye-Sung; Park, Hye-Mi; Kim, Chang-Hyun; Kim, Tai-Gyu

    2011-11-03

    Protein vaccines may be a useful strategy for cancer immunotherapy because recombinant tumor antigen proteins can be produced on a large scale at relatively low cost and have been shown to be safe for clinical application. However, protein vaccines have historically exhibited poor immunogenicity; thus, an improved strategy is needed for successful induction of immune responses. TAT peptide is a protein transduction domain composed of an 11-amino acid peptide (TAT(47-57): YGRKKRRQRRR). The positive charge of this peptide allows protein antigen fused with it to improve cell penetration. Poly(I:C) is a synthetic double-stranded RNA that is negatively charged and favors interaction with the cationic TAT peptide. Poly(I:C) has been reported on adjuvant role in tumor vaccine through promotion of immune responses. Therefore, we demonstrated that vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) can induce anti-tumor immunity in a murine colorectal tumor model. Splenocytes from mice vaccinated with a mixture of TAT-CEA fusion protein and poly(I:C) effectively induced CEA-specific IFN-γ-producing T cells and showed cytotoxic activity specific for MC-38-cea2 tumor cells expressing CEA. Vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) delayed tumor growth in MC-38-cea-2 tumor-bearing mice. Depletion of CD8(+) T cells and NK cells reversed the inhibition of tumor growth in an MC-38-cea2-bearing mice, indicating that CD8(+) T cells and NK cells are responsible for anti-tumor immunity by vaccine with a mixture of TAT-CEA fusion protein and poly(I:C). Taken together, these results suggest that poly(I:C) could be used as a potent adjuvant to induce the anti-tumor immunity of a TAT-CEA fusion protein vaccine in a murine colorectal tumor model. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Antitumour effects of PLC-gamma1-(SH2)2-TAT fusion proteins on EGFR/c-erbB-2-positive breast cancer cells.

    PubMed

    Katterle, Y; Brandt, B H; Dowdy, S F; Niggemann, B; Zänker, K S; Dittmar, T

    2004-01-12

    Due to its pivotal role in the growth factor-mediated tumour cell migration, the adaptor protein phospholipase C-gamma1 (PLC-gamma1) is an appropriate target to block ultimately the spreading of EGFR/c-erbB-2-positive tumour cells, thereby minimising metastasis formation. Here, we present an approach to block PLC-gamma1 activity by using protein-based PLC-gamma1 inhibitors consisting of PLC-gamma1 SH2 domains, which were fused to the TAT-transduction domain to ensure a high protein transduction efficiency. Two proteins were generated containing one PLC-gamma1-SH2-domain (PS1-TAT) or two PLC-gamma1-SH2 domains (PS2-TAT). PS2-TAT treatment of the EGFR/c-erbB-2-positive cell line MDA-HER2 resulted in a reduction of the EGF-mediated PLC-gamma1 tyrosine phosphorylation of about 30%, concomitant with a complete abrogation of the EGF-driven calcium influx. In addition to this, long-term PS2-TAT treatment both reduces the EGF-mediated migration of about 75% combined with a markedly decreased time locomotion of single MDA-HER2 cells as well as decreases the proliferation of MDA-HER2 cells by about 50%. Due to its antitumoral capacity on EGFR/c-erbB-2-positive breast cancer cells, we conclude from our results that the protein-based PLC-gamma1 inhibitor PS2-TAT may be a means for novel adjuvant antitumour strategies to minimise metastasis formation because of the blockade of cell migration and proliferation.

  18. Combination with anti-tit-for-tat remedies problems of tit-for-tat.

    PubMed

    Yi, Su Do; Baek, Seung Ki; Choi, Jung-Kyoo

    2017-01-07

    One of the most important questions in game theory concerns how mutual cooperation can be achieved and maintained in a social dilemma. In Axelrod's tournaments of the iterated prisoner's dilemma, Tit-for-Tat (TFT) demonstrated the role of reciprocity in the emergence of cooperation. However, the stability of TFT does not hold in the presence of implementation error, and a TFT population is prone to neutral drift to unconditional cooperation, which eventually invites defectors. We argue that a combination of TFT and anti-TFT (ATFT) overcomes these difficulties in a noisy environment, provided that ATFT is defined as choosing the opposite to the opponent's last move. According to this TFT-ATFT strategy, a player normally uses TFT; turns to ATFT upon recognizing his or her own error; returns to TFT either when mutual cooperation is recovered or when the opponent unilaterally defects twice in a row. The proposed strategy provides simple and deterministic behavioral rules for correcting implementation error in a way that cannot be exploited by the opponent, and suppresses the neutral drift to unconditional cooperation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

    PubMed

    Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

    2018-02-01

    New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  20. PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

    PubMed

    Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

    2017-05-01

    Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO 2 ) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO 2 . We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

  1. Tit for tat in heterogeneous populations

    NASA Astrophysics Data System (ADS)

    Nowak, Martin A.; Sigmund, Karl

    1992-01-01

    THE 'iterated prisoner's dilemma' is now the orthodox paradigm for the evolution of cooperation among selfish individuals. This viewpoint is strongly supported by Axelrod's computer tournaments, where 'tit for tat' (TFT) finished first1. This has stimulated interest in the role of reciprocity in biological societies1-8. Most theoretical investigations, however, assumed homogeneous populations (the setting for evolutionary stable strategies9,10) and programs immune to errors. Here we try to come closer to the biological situation by following a program6 that takes stochasticities into account and investigates representative samples. We find that a small fraction of TFT players is essential for the emergence of reciprocation in a heterogeneous population, but only paves the way for a more generous strategy. TFT is the pivot, rather than the aim, of an evolution towards cooperation.

  2. Tat-modified leptin is more accessible to hypothalamus through brain-blood barrier with a significant inhibition of body-weight gain in high-fat-diet fed mice.

    PubMed

    Zhang, C; Su, Z; Zhao, B; Qu, Q; Tan, Y; Cai, L; Li, X

    2010-01-01

    Obesity in human was found mainly due to the poor transportation of leptin through brain-blood barrier (BBB), called as leptin resistance. To produce a leptin capable of penetrating BBB, we have added Tat-PTD(9) to the C terminal of leptin to construct a fusion protein. The fusion Tat-leptin and native leptin genes were synthesized by single-step insertion of a polymerase chain reaction and expressed in Escherichia coli BL21 (Rosseta). The expressing products were purified and renatured by Ni-NTA affinity chromatography, and identified by the molecular size in SDS-PAGE gel and by its immunoreactivity to specific antibody with Western-blotting assay. To bio-functionally evaluate the fusion protein, Balb/c mice fed with high-fat diet (HFD) were given Tat-leptin, leptin or saline for 19 days. The immunohistochemical staining showed the increases in positive stains for the leptin in the region of hypothalamus of the HFD mice with either Tat-leptin or leptin as compared to saline group, but the staining intensity and frequency in the group with Tat-leptin were stronger and higher than those in the group with leptin. Furthermore, the most efficiency in preventing the body-weight gain caused by HFD was found in Tat-leptin group among these three groups. These results suggest that Tat-modified leptin may become a great potential candidate for the prevention or therapy of obese patients. J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart , New York.

  3. A novel branched TAT(47-57) peptide for selective Ni(2+) introduction into the human fibrosarcoma cell nucleus.

    PubMed

    Szyrwiel, Łukasz; Shimura, Mari; Shirataki, Junko; Matsuyama, Satoshi; Matsunaga, Akihiro; Setner, Bartosz; Szczukowski, Łukasz; Szewczuk, Zbigniew; Yamauchi, Kazuto; Malinka, Wiesław; Chavatte, Laurent; Łobinski, Ryszard

    2015-07-01

    A TAT47-57 peptide was modified on the N-terminus by elongation with a 2,3-diaminopropionic acid residue and then by coupling of two histidine residues on its N-atoms. This branched peptide could bind to Ni under physiological conditions as a 1 : 1 complex. We demonstrated that the complex was quantitatively taken up by human fibrosarcoma cells, in contrast to Ni(2+) ions. Ni localization (especially at the nuclei) was confirmed by imaging using both scanning X-ray fluorescence microscopy and Newport Green fluorescence. A competitive assay with Newport Green showed that the latter displaced the peptide ligand from the Ni-complex. Ni(2+) delivered as a complex with the designed peptide induced substantially more DNA damage than when introduced as a free ion. The availability of such a construct opens up the way to investigate the importance of the nucleus as a target for the cytotoxicity, genotoxicity or carcinogenicity of Ni(2+).

  4. Situation selection is a particularly effective emotion regulation strategy for people who need help regulating their emotions.

    PubMed

    Webb, Thomas L; Lindquist, Kristen A; Jones, Katelyn; Avishai, Aya; Sheeran, Paschal

    2018-03-01

    Situation selection involves choosing situations based on their likely emotional impact and may be less cognitively taxing or challenging to implement compared to other strategies for regulating emotion, which require people to regulate their emotions "in the moment"; we thus predicted that individuals who chronically experience intense emotions or who are not particularly competent at employing other emotion regulation strategies would be especially likely to benefit from situation selection. Consistent with this idea, we found that the use of situation selection interacted with individual differences in emotional reactivity and competence at emotion regulation to predict emotional outcomes in both a correlational (Study 1; N = 301) and an experimental field study (Study 2; N = 125). Taken together, the findings suggest that situation selection is an effective strategy for regulating emotions, especially for individuals who otherwise struggle to do so.

  5. Penetration of HIV-1 Tat47-57 into PC/PE Bilayers Assessed by MD Simulation and X-ray Scattering.

    PubMed

    Neale, Chris; Huang, Kun; García, Angel E; Tristram-Nagle, Stephanie

    2015-09-22

    The interactions of the basic, cell-penetrating region (Y47GRKKRRQRRR57) of the HIV-1 Tat protein with dioleoylphosphatidylcholine (DOPC) bilayers were previously assessed by comparing experimental X-ray diffuse scattering with atomistic molecular dynamics simulations. Here, we extend this investigation by evaluating the influence of phosphatidylethanolamine (PE) lipids. Using experimental bilayer form factors derivedfrom X-ray diffuse scattering data as a guide, our simulations indicate that Tat peptides localize close to the carbonyl-glycerol group in the headgroup region of bilayers composed of either DOPC or DOPC:DOPE (1:1) lipid. Our results also suggest that Tat peptides may more frequently insert into the hydrophobic core of bilayers composed of PC:PE (1:1) lipids than into bilayers composed entirely of PC lipids. PE lipids may facilitate peptide translocation across a lipid bilayer by stabilizing intermediate states in which hydrated peptides span the bilayer.

  6. The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner

    PubMed Central

    Puyal, Julien; Margue, Christiane; Michel, Sébastien; Kreis, Stephanie; Kulms, Dagmar; Barras, David; Nahimana, Aimable; Widmann, Christian

    2016-01-01

    Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment. PMID:27602963

  7. Homogenizing microwave illumination in thermoacoustic tomography by a linear-to-circular polarizer based on frequency selective surfaces

    NASA Astrophysics Data System (ADS)

    He, Yu; Shen, Yuecheng; Feng, Xiaohua; Liu, Changjun; Wang, Lihong V.

    2017-08-01

    A circularly polarized antenna, providing more homogeneous illumination compared to a linearly polarized antenna, is more suitable for microwave induced thermoacoustic tomography (TAT). The conventional realization of a circular polarization is by using a helical antenna, but it suffers from low efficiency, low power capacity, and limited aperture in TAT systems. Here, we report an implementation of a circularly polarized illumination method in TAT by inserting a single-layer linear-to-circular polarizer based on frequency selective surfaces between a pyramidal horn antenna and an imaging object. The performance of the proposed method was validated by both simulations and experimental imaging of a breast tumor phantom. The results showed that a circular polarization was achieved, and the resultant thermoacoustic signal-to-noise was twice greater than that in the helical antenna case. The proposed method is more desirable in a waveguide-based TAT system than the conventional method.

  8. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    DOE PAGES

    Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; ...

    2017-07-24

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  9. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

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

    Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas thosemore » expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Secdependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. But, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Thus, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  10. Mutations at Tyrosine 88, Lysine 92 and Tyrosine 470 of human dopamine transporter result in an attenuation of HIV-1 Tat-induced inhibition of dopamine transport

    PubMed Central

    Midde, Narasimha M.; Yuan, Yaxia; Quizon, Pamela M.; Sun, Wei-Lun; Huang, Xiaoqin; Zhan, Chang-Guo; Zhu, Jun

    2015-01-01

    HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [3H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [3H]DA uptake and [3H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [3H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [3H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding. PMID:25604666

  11. Situation Selection and Modification for Emotion Regulation in Younger and Older Adults.

    PubMed

    Livingstone, Kimberly M; Isaacowitz, Derek M

    2015-11-01

    This research investigated age differences in use and effectiveness of situation selection and situation modification for emotion regulation. Socioemotional selectivity theory suggests stronger emotional well-being goals in older age; emotion regulation may support this goal. Younger and older adults assigned to an emotion regulation or "just view" condition first freely chose to engage with negative, neutral, or positive material (situation selection), then chose to view or skip negative and positive material (situation modification), rating affect after each experience. In both tasks, older adults in both goal conditions demonstrated pro-hedonic emotion regulation, spending less time with negative material compared to younger adults. Younger adults in the regulate condition also engaged in pro-hedonic situation selection, but not modification. Whereas situation selection was related to affect, modification of negative material was not. This research supports more frequent pro-hedonic motivation in older age, as well as age differences in use of early-stage emotion regulation.

  12. Situation Selection and Modification for Emotion Regulation in Younger and Older Adults

    PubMed Central

    Livingstone, Kimberly M.; Isaacowitz, Derek M.

    2016-01-01

    This research investigated age differences in use and effectiveness of situation selection and situation modification for emotion regulation. Socioemotional selectivity theory suggests stronger emotional well-being goals in older age; emotion regulation may support this goal. Younger and older adults assigned to an emotion regulation or “just view” condition first freely chose to engage with negative, neutral, or positive material (situation selection), then chose to view or skip negative and positive material (situation modification), rating affect after each experience. In both tasks, older adults in both goal conditions demonstrated pro-hedonic emotion regulation, spending less time with negative material compared to younger adults. Younger adults in the regulate condition also engaged in pro-hedonic situation selection, but not modification. Whereas situation selection was related to affect, modification of negative material was not. This research supports more frequent pro-hedonic motivation in older age, as well as age differences in use of early-stage emotion regulation. PMID:26998196

  13. Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

    PubMed Central

    Chun, R; Glabe, C G; Fan, H

    1990-01-01

    Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis. Images PMID:2186178

  14. A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

    PubMed

    Bozue, Joel; Cote, Christopher K; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J; Kijek, Todd K; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G; Bell, Todd; Worsham, Patricia

    2014-01-01

    Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

  15. A Yersinia pestis tat Mutant Is Attenuated in Bubonic and Small-Aerosol Pneumonic Challenge Models of Infection but Not As Attenuated by Intranasal Challenge

    PubMed Central

    Bozue, Joel; Cote, Christopher K.; Chance, Taylor; Kugelman, Jeffrey; Kern, Steven J.; Kijek, Todd K.; Jenkins, Amy; Mou, Sherry; Moody, Krishna; Fritz, David; Robinson, Camenzind G.; Bell, Todd; Worsham, Patricia

    2014-01-01

    Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge. PMID:25101850

  16. The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study

    PubMed Central

    2014-01-01

    Background Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. Findings Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. Conclusions Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. PMID:24961156

  17. Evidence that a sequence similar to TAR is important for induction of the JC virus late promoter by human immunodeficiency virus type 1 Tat.

    PubMed Central

    Chowdhury, M; Taylor, J P; Chang, C F; Rappaport, J; Khalili, K

    1992-01-01

    A specific RNA sequence located in the leader of all human immunodeficiency virus type 1 (HIV-1) mRNAs termed the transactivation response element, or TAR, is a primary target for induction of HIV-1 long terminal repeat activity by the HIV-1-derived trans-regulatory protein, Tat. Human neurotropic virus, JC virus (JCV), a causative agent of the degenerative demyelinating disease progressive multifocal leukoencephalopathy, contains sequences in the 5' end of the late RNA species with an extensive homology to HIV-1 TAR. In this study, we examined the possible role of the JCV-derived TAR-homologous sequence in Tat-mediated activation of the JCV late promoter (Tada et al., Proc. Natl. Acad. Sci. USA 87:3479-3483, 1990). Results from site-directed mutagenesis revealed that critical G residues required for the function of HIV-1 TAR that are conserved in the JCV TAR homolog play an important role in Tat activation of the JCV promoter. In addition, in vivo competition studies suggest that shared regulatory components mediate Tat activation of the JCV late and HIV-1 long terminal repeat promoters. Furthermore, we showed that the JCV-derived TAR sequence behaves in the same way as HIV-1 TAR in response to two distinct Tat mutants, one of which that has no ability to bind to HIV-1 TAR and another that lacks transcriptional activity on a responsive promoter. These results suggest that the TAR homolog of the JCV late promoter is responsive to HIV-1 Tat induction and thus may participate in the overall activation of the JCV late promoter mediated by this transactivation. Images PMID:1331525

  18. In Vivo MR Imaging of Glioma Recruitment of Adoptive T-Cells Labeled with NaGdF4 -TAT Nanoprobes.

    PubMed

    Zhang, Hua; Wu, Yue; Wang, Jing; Tang, Zhongmin; Ren, Yan; Ni, Dalong; Gao, Hongbo; Song, Ruixue; Jin, Teng; Li, Qiao; Bu, Wenbo; Yao, Zhenwei

    2018-01-01

    Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T-cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T-cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T-cell immunotherapy targeting glioma. In this work, ultrasmall T 1 MR-based nanoprobes, NaGdF 4 -TAT, as molecular probes with high longitudinal relaxivity (8.93 mm -1 s -1 ) are designed. By means of HIV-1 transactivator (TAT) peptides, nearly 95% of the adoptive T-cells are labeled with the NaGdF 4 -TAT nanoprobes without any measurable side effects on the labeled T-cells, which is remarkably superior to that of the control fluorescein isothiocyanate-NaGdF 4 concerning labeling efficacy. Labeled adoptive T-cell clusters can be sensitively tracked in an orthotopic GL261-glioma model 24 h after intravenous infusion of 10 7 labeled T-cells by T 1 -weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF 4 -TAT nanoprobes labeling of T-cells may be a promising method to track adoptive T-cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

    PubMed Central

    Yan, Dong; Perriman, Rhonda; Igel, Haller; Howe, Kenneth J.; Neville, Megan; Ares, Manuel

    1998-01-01

    A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells. PMID:9710584

  20. Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²⁺ overload.

    PubMed

    Fitting, Sylvia; Knapp, Pamela E; Zou, Shiping; Marks, William D; Bowers, M Scott; Akbarali, Hamid I; Hauser, Kurt F

    2014-09-17

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuro

  1. Essentials of TAT and Other Storytelling Techniques Assessment. Essentials of Psychological Assessment Series.

    ERIC Educational Resources Information Center

    Teglasi, Hedwig

    This book provides guidance into the use of storytelling techniques as an approach to personality assessment and explains how to administer, score, and interpret such tests. The tests discussed include the Thematic Apperception Test (TAT), the Roberts Apperception Test for Children, and the TEMAS (Tell-Me-a-Story). Each chapter contains callout…

  2. Information security for compliance with select agent regulations.

    PubMed

    Lewis, Nick; Campbell, Mark J; Baskin, Carole R

    2015-01-01

    The past decade has seen a significant rise in research on high-consequence human and animal pathogens, many now known as "select agents." While physical security around these agents is tightly regulated, information security standards are still lagging. The understanding of the threats unique to the academic and research environment is still evolving, in part due to poor communication between the various stakeholders. Perhaps as a result, information security guidelines published by select agent regulators lack the critical details and directives needed to achieve even the lowest security level of the Federal Information Security Management Act (FISMA). While only government agencies are currently required to abide by the provisions of FISMA (unless specified as preconditions for obtaining government grants or contracts--still a relatively rare or narrowly scoped occurrence), the same strategies were recently recommended by executive order for others. We propose that information security guidelines for select agent research be updated to promulgate and detail FISMA standards and processes and that the latter be ultimately incorporated into select agent regulations. We also suggest that information security in academic and research institutions would greatly benefit from active efforts to improve communication among the biosecurity, security, and information technology communities, and from a secure venue for exchange of timely information on emerging threats and solutions in the research environment.

  3. Information Security for Compliance with Select Agent Regulations

    PubMed Central

    Lewis, Nick; Campbell, Mark J.

    2015-01-01

    The past decade has seen a significant rise in research on high-consequence human and animal pathogens, many now known as “select agents.” While physical security around these agents is tightly regulated, information security standards are still lagging. The understanding of the threats unique to the academic and research environment is still evolving, in part due to poor communication between the various stakeholders. Perhaps as a result, information security guidelines published by select agent regulators lack the critical details and directives needed to achieve even the lowest security level of the Federal Information Security Management Act (FISMA). While only government agencies are currently required to abide by the provisions of FISMA (unless specified as preconditions for obtaining government grants or contracts—still a relatively rare or narrowly scoped occurrence), the same strategies were recently recommended by executive order for others. We propose that information security guidelines for select agent research be updated to promulgate and detail FISMA standards and processes and that the latter be ultimately incorporated into select agent regulations. We also suggest that information security in academic and research institutions would greatly benefit from active efforts to improve communication among the biosecurity, security, and information technology communities, and from a secure venue for exchange of timely information on emerging threats and solutions in the research environment. PMID:26042864

  4. Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

    PubMed Central

    Maubert, Monique E.; Pirrone, Vanessa; Rivera, Nina T.; Wigdahl, Brian; Nonnemacher, Michael R.

    2016-01-01

    In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS). Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART), CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND). A number of HIV-1 proteins (Tat, gp120, Nef, Vpr) have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients. PMID:26793168

  5. Herpes Simplex Virus Type 2 Triggers Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency and Collaborates with HIV-1 Tat

    PubMed Central

    Zhu, Xiaolei; Ma, Xinting; Yan, Qin; Zeng, Yi; Guo, Yuanyuan; Feng, Ninghan; Lu, Chun

    2012-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for Kaposi's sarcoma (KS) development without other cofactors. Previously, we identified that both human immunodeficiency type 1 (HIV-1) Tat and herpes simplex virus 1 (HSV-1) were important cofactors reactivating KSHV from latency. Here, we further investigated the potential of herpes simplex virus 2 (HSV-2) to influence KSHV replication and examined the role of Tat in this procedure. We demonstrated that HSV-2 was a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV Rta and a luciferase reporter assay testing Rta promoter-driven luciferase activity. Mechanistic studies showed that HSV-2 infection activated nuclear factor-kappa B (NF-κB) signaling pathway. Inhibition of NF-κB pathway enhanced HSV-2-mediated KSHV activation, whereas activation of NF-κB pathway suppressed KSHV replication in HSV-2-infected BCBL-1 cells. Additionally, ectopic expression of Tat enhanced HSV-2-induced KSHV replication. These novel findings suggest a role of HSV-2 in the pathogenesis of KS and provide the first laboratory evidence that Tat may participate HSV-2-mediated KSHV activation, implying the complicated pathogenesis of acquired immunodeficiency syndrome (AIDS)-related KS (AIDS-KS) patients. PMID:22347501

  6. Using the TAT to Assess the Relation Between Gender Identity and the Use of Defense Mechanisms.

    PubMed

    Cramer, Phebe

    2017-01-01

    The purpose of this study is to explore whether 2 different dimensions of personality, when assessed at an implicit level with the Thematic Apperception Test (TAT; Murray, 1943 ) will show a theoretically meaningful coherence not demonstrated when 1 is assessed at an implicit level and the other at an explicit level. Gender identity and defense mechanisms were assessed implicitly using the TAT. Gender identity was compared with a self-report measure of gender-related attributes assessed at the explicit level. The results showed a theoretically meaningful coherence when different dispositions were assessed at the same level, but a lack of agreement when similar dispositions were assessed at different levels. The study is based on a secondary analysis of data from 2 previously published papers (Cramer, 1998 ; Cramer & Westergren, 1999 ).

  7. The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

    PubMed

    Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

    2017-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se . Instead

  8. The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    PubMed Central

    Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

    2017-01-01

    The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

  9. 78 FR 56234 - Multi-Agency Informational Meeting Concerning Compliance with the Select Agent Regulations...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-12

    ... Informational Meeting Concerning Compliance with the Select Agent Regulations; Public Webcast AGENCY: Centers... purpose of the webcast is to provide guidance related to the select agent regulations established under... Justice Information Services. Changes to Section 11(Security) of the select agent regulations including...

  10. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    PubMed

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  11. Fluctuations in Tat copy number when it counts the most: a possible mechanism to battle the HIV latency

    PubMed Central

    2013-01-01

    The HIV-1 virus can enter a dormant state and become inactive, which reduces accessibility by antiviral drugs. We approach this latency problem from an unconventional point of view, with the focus on understanding how intrinsic chemical noise (copy number fluctuations of the Tat protein) can be used to assist the activation process of the latent virus. Several phase diagrams have been constructed in order to visualize in which regions of the parameter space noise can drive the activation process. Essential to the study is the use of a hyperbolic coordinate system, which greatly facilitates quantification of how the various reaction rate combinations shape the noise behavior of the Tat protein feedback system. We have designed a mathematical manual of how to approach the problem of activation quantitatively, and introduce the notion of an “operating point” of the virus. For both noise-free and noise-based strategies we show how operating point off-sets induce changes in the number of Tat molecules. The major result of the analysis is that for every noise-free strategy there is a noise-based strategy that requires lower dosage, but achieves the same anti-latency effect. It appears that the noise-based activation is advantageous for every operating point. PMID:23497153

  12. A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

    PubMed Central

    2014-01-01

    Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

  13. An attenuated herpes simplex virus type 1 (HSV1) encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

    PubMed

    Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C

    2014-01-01

    Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

  14. An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

    PubMed Central

    Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C.

    2014-01-01

    Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and

  15. TRIM-directed selective autophagy regulates immune activation.

    PubMed

    Kimura, Tomonori; Jain, Ashish; Choi, Seong Won; Mandell, Michael A; Johansen, Terje; Deretic, Vojo

    2017-05-04

    Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.

  16. TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

    PubMed

    Erlich-Hadad, Tal; Hadad, Rita; Feldman, Anat; Greif, Hagar; Lictenstein, Michal; Lorberboum-Galski, Haya

    2018-03-01

    Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Morphological, Histochemical, Immunohistochemical, and Ultrastructural Characterization of Tumors and Dysplastic and Non-Neoplastic Lesions Arising in BK Virus/tat Transgenic Mice

    PubMed Central

    Altavilla, Giuseppe; Trabanelli, Cecilia; Merlin, Michela; Caputo, Antonella; Lanfredi, Massimo; Barbanti-Brodano, Giuseppe; Corallini, Alfredo

    1999-01-01

    To study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS. PMID:10233861

  18. Metabolic fuels: regulating fluxes to select mix.

    PubMed

    Weber, Jean-Michel

    2011-01-15

    Animals must regulate the fluxes of multiple fuels to support changing metabolic rates that result from variation in physiological circumstances. The aim of fuel selection strategies is to exploit the advantages of individual substrates while minimizing the impact of disadvantages. All exercising mammals share a general pattern of fuel selection: at the same %V(O(2,max)) they oxidize the same ratio of lipids to carbohydrates. However, highly aerobic species rely more on intramuscular fuels because energy supply from the circulation is constrained by trans-sarcolemmal transfer. Fuel selection is performed by recruiting different muscles, different fibers within the same muscles or different pathways within the same fibers. Electromyographic analyses show that shivering humans can modulate carbohydrate oxidation either through the selective recruitment of type II fibers within the same muscles or by regulating pathway recruitment within type I fibers. The selection patterns of shivering and exercise are different: at the same %V(O(2,max)), a muscle producing only heat (shivering) or significant movement (exercise) strikes a different balance between lipid and carbohydrate oxidation. Long-distance migrants provide an excellent model to characterize how to increase maximal substrate fluxes. High lipid fluxes are achieved through the coordinated upregulation of mobilization, transport and oxidation by activating enzymes, lipid-solubilizing proteins and membrane transporters. These endurance athletes support record lipolytic rates in adipocytes, use lipoprotein shuttles to accelerate transport and show increased capacity for lipid oxidation in muscle mitochondria. Some migrant birds use dietary omega-3 fatty acids as performance-enhancing agents to boost their ability to process lipids. These dietary fatty acids become incorporated in membrane phospholipids and bind to peroxisome proliferator-activated receptors to activate membrane proteins and modify gene expression.

  19. [Critical considerations on the legal regulation of sex selection (Part I)].

    PubMed

    Pérez Alonso, Esteban Juan

    2002-01-01

    Gender selection, and particularly its regulation, is a controversial issue. The author discusses the current problems surrounding gender selection from the very beginnings, and illustrates his views with an actual and controversial case in which a woman allowed to undergo artificial insemination was given the possibility of choosing the sex of her child. The author also discusses possible solutions and the penal, administrative regulation of the issue, as well as examining the court's decision in this particular case.

  20. Tat-Mediated Induction of miRs-34a & -138 Promotes Astrocytic Activation via Downregulation of SIRT1: Implications for Aging in HAND.

    PubMed

    Hu, Guoku; Liao, Ke; Yang, Lu; Pendyala, Gurudutt; Kook, Yeonhee; Fox, Howard S; Buch, Shilpa

    2017-09-01

    Astrocyte activation is a hallmark of HIV infection and aging in the CNS. In chronically infected HIV patients, prolonged activation of astrocytes has been linked to accelerated aging including but not limited to neurocognitive impairment and frailty. The current study addresses the role of HIV protein Tat in inducing a set of small noncoding microRNAs (miRNA) that play critical role in astrogliosis. In our efforts to link astrocyte activation as an indicator of aging, we assessed the brains of both wild type and HIV transgenic rats for the expression of glial fibrillary acidic protein (GFAP). As expected, in the WT animals we observed age-dependent increase in astrogliosis in the older animals compared to the younger group. Interestingly, compared to the young WT group, young HIV Tg rats exhibited higher levels of GFAP in this trend was also observed in the older HIV Tg rats compared to the older WT group. Based on the role of SIRT1 in aging and the regulation of SIRT1 by miRNAs-34a and -138, we next assessed the expression levels of these miRs in the brains of both the young an old WT and HIV Tg rats. While there were no significant differences in the young WT versus the HIV Tg rats, in the older HIV Tg rats there was a significant upregulation in the expression of miRs-34a & -138 in the brains. Furthermore, increased expression of miRs-34a & -138 in the older Tg rats, correlated with a concomitant decrease in their common anti-aging target protein SIRT1, in the brains of these animals. To delineate the mechanism of action we assessed the role of HIV-Tat (present in the Tg rats) in inducing miRs-34a & -138 in both the primary astrocytes and the astrocytoma cell line A172, thereby leading to posttranscriptional suppression of SIRT1 with a concomitant up regulation of NF-kB driven expression of GFAP.

  1. pH-Sensitive nanoparticles as smart carriers for selective intracellular drug delivery to tumor.

    PubMed

    Li, Xin-Xin; Chen, Jing; Shen, Jian-Min; Zhuang, Ran; Zhang, Shi-Qi; Zhu, Zi-Yun; Ma, Jing-Bo

    2018-05-05

    Herein, a smart pH-sensitive nanoparticle (DGL-PEG-Tat-KK-DMA-DOX) was prepared to achieve the selective intracellular drug delivery. In this nanoparticle, a PEG-grafted cell penetrating peptide (PEG-Tat-KK) was designed and acted as the cell penetrating segment. By introducing the pH-sensitive amide bonds between the peptide and blocking agent (2,3-dimethylmaleic anhydride, DMA), the controllable moiety (PEG-Tat-KK-DMA) endowed the nanoparticle with a charge-switchable shell and temporarily blocked penetrating function, thus improving the specific internalization. Besides, dendrigraft poly-L-lysine (DGL) used as the skeleton can greatly improve the drug loading because of the highly dendritic framework. Under the stimuli of acidic pH, this nanoparticle exhibited a remarkable charge-switchable property. The drug release showed an expected behavior with little release in the neutral pH media but relatively fast release in the acidic media. The in vitro experiments revealed that the cellular uptake and cytotoxicity were significantly enhanced after the pH was decreased. In vivo biodistribution and antitumor research indicated that the nanoparticle had noteworthy specificity and antitumor efficacy with a tumor inhibition rate of 79.7%. These results verified this nanoparticle could efficiently improve the selective intracellular delivery and possessed a great potential in tumor treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Therapeutic Immunization with HIV-1 Tat Reduces Immune Activation and Loss of Regulatory T-Cells and Improves Immune Function in Subjects on HAART

    PubMed Central

    Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J.; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S.; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

    2010-01-01

    Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4+ T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4+ and CD8+ cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4+ T cells and B cells with reduction of CD8+ T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4+ and CD8+ T cells were accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the

  3. Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART.

    PubMed

    Ensoli, Barbara; Bellino, Stefania; Tripiciano, Antonella; Longo, Olimpia; Francavilla, Vittorio; Marcotullio, Simone; Cafaro, Aurelio; Picconi, Orietta; Paniccia, Giovanni; Scoglio, Arianna; Arancio, Angela; Ariola, Cristina; Ruiz Alvarez, Maria J; Campagna, Massimo; Scaramuzzi, Donato; Iori, Cristina; Esposito, Roberto; Mussini, Cristina; Ghinelli, Florio; Sighinolfi, Laura; Palamara, Guido; Latini, Alessandra; Angarano, Gioacchino; Ladisa, Nicoletta; Soscia, Fabrizio; Mercurio, Vito S; Lazzarin, Adriano; Tambussi, Giuseppe; Visintini, Raffaele; Mazzotta, Francesco; Di Pietro, Massimo; Galli, Massimo; Rusconi, Stefano; Carosi, Giampiero; Torti, Carlo; Di Perri, Giovanni; Bonora, Stefano; Ensoli, Fabrizio; Garaci, Enrico

    2010-11-11

    Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent

  4. Labeling Efficacy of Superparamagnetic Iron Oxide Nanoparticles to Human Neural Stem Cells: Comparison of Ferumoxides, Monocrystalline Iron Oxide, Cross-linked Iron Oxide (CLIO)-NH2 and tat-CLIO

    PubMed Central

    Song, Miyeoun; Kim, Yunhee; Lim, Dongyeol; Song, In-Chan; Yoon, Byung-Woo

    2007-01-01

    Objective We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH2 and tat-CLIO. Materials and Methods The hNSCs (5 × 105 HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 µg/ml of ferumoxides, MION or CLIO-NH2, and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. Results The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15 ± 0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH2, respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH2 into the hNSCs was comparable to that of tat-CLIO. Conclusion For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH2 and the transfection agent PLL. PMID:17923778

  5. Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-tat-based vaccine candidate to elicit cellular immunity in BALB/c mice.

    PubMed

    Panahi, Zeinab; Abdoli, Asghar; Mosayebi, Ghasem; Mahdavi, Mehdi; Bahrami, Fariborz

    2018-03-01

    To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model. Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG 2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05). Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

  6. Mapping the architecture of the HIV-lTat circuit: A decision-making circuit that lacks bistability and exploits stochastic noise

    PubMed Central

    Razooky, Brandon S.; Weinberger, Leor S.

    2014-01-01

    Upon infection of a CD4+ T cell, HIV-l appears to ‘choose’ between two alternate fates: active replication or a long-lived dormant statetermed proviral latency. A transcriptional positive-feedback loop generated by the HIV-l Tat protein appears sufficient to mediate this decision. Here, we describea coupled wet-lab and computational approach that uses mathematical modeling and live-cell time-lapse microscopy to map the architecture of the HIV-l Tat transcriptional regulatorycircuit and generate predictive models of HIV-l latency. This approach provided the first characterization of a ‘decision-making’ circuit that lacks bistability andinstead exploits stochastic fluctuations in cellular molecules (i.e. noise) to generate a decision between an on or off transcriptional state. PMID:21167940

  7. Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

    PubMed

    Ul Ain, Qurrat; Lee, Jong Hwan; Woo, Young Sun; Kim, Yong-Hee

    2016-09-01

    Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

  8. Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43.

    PubMed

    Heyman, Nathanael S; Kurjiaka, David T; Ek Vitorin, Jose F; Burt, Janis M

    2009-07-01

    Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

  9. The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

    PubMed Central

    Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

    2011-01-01

    Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

  10. TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

    PubMed Central

    Free, Paul; Lévy, Raphaël

    2015-01-01

    The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles. PMID:25836335

  11. Maesopsin 4-O-beta-D-glucoside, a natural compound isolated from the leaves of Artocarpus tonkinensis, inhibits proliferation and up-regulates HMOX1, SRXN1 and BCAS3 in acute myeloid leukemia.

    PubMed

    Pozzesi, N; Pierangeli, S; Vacca, C; Falchi, L; Pettorossi, V; Martelli, M P; Thuy, T T; Ninh, P T; Liberati, A M; Riccardi, C; Sung, T V; Delfino, D V

    2011-06-01

    The leaves of Artocarpus tonkinensis are used in Vietnamese traditional medicine for treatment of arthritis, and the compound maesopsin 4-O-β-D-glucoside (TAT-2), isolated from them, inhibits the proliferation of activated T cells. Our goal was to test the anti-proliferative activity of TAT-2 on the T-cell leukemia, Jurkat, and on the acute myeloid leukemia, OCI-AML. TAT-2 inhibited the growth of OCI-AML (and additional acute myeloid leukemia cells) but not Jurkat cells. Growth inhibition was shown to be due to inhibition of proliferation rather than increase in cell death. Analysis of cytokine release showed that TAT-2 stimulated the release of TGF-β, yet TGF-β neutralization did not reverse the maesopsin-dependent effect. Gene expression profiling determined that maesopsin modulated 19 identifiable genes. Transcription factor CP2 was the gene most significantly modulated. Real-time PCR validated that up-regulation of sulphiredoxin 1 homolog (SRXN1), hemeoxygenase 1 (HMOX1), and breast carcinoma amplified sequence 3 (BCAS3) were consistently modulated.

  12. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

    PubMed

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

  13. Imaging HIV-1 Tat Trafficking and Interactions by Engineered Green-Fluorescent-Protein Tagging

    NASA Astrophysics Data System (ADS)

    Beltram, Fabio

    2002-03-01

    The direct monitoring of protein function in live cells under physiologically relevant conditions is one of the most powerful and innovative methodologies for proteomics. Efficient florescent probes fully compatible with human-cell expression are the fundamental tools for these studies and their optimization opens the way to resolution at the single-protein level. Biological events involving protein pairs are also directly accessible thanks to tuning of protein-tag spectral properties and production of complementary pairs. Such pairs are characterized by overlapping absorption (for the acceptor tag) and emission (for the donor tag) spectra. By tagging the proteins of interest with acceptor and donor molecules, protein interaction can be directly visualized by FRET, fluorescent resonant energy transfer. In this talk we shall present the design by molecular dynamics calculations and the application of optimized green fluorescent proteins to the study of the human immunodeficiency virus HIV-1 proteomics. In particular trafficking and cellular interactions of HIV-1 transactivator protein Tat in live human cells will be presented. Tat localization and complex internalization pathways of exogenous molecules will be presented thanks to the peculiar optical properties of mutated GFPs. Cellular protein partners and subcellular interaction sites will be identified and directly visualized. The relevance of such results and of advanced spectroscopic and imaging techniques for a new level of understanding of biological processes and its significance for advancement in molecular biology will be underlined. A. Marcello et al., J. Biol. Chem. 276, 39220 (2001). R. Cinelli et al., Appl. Phys. Lett. 79, 3353 (2001).

  14. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

    PubMed Central

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285

  15. Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

    PubMed Central

    Dalvi, Pranjali; Sharma, Himanshu; Chinnappan, Mahendran; Sanderson, Miles; Allen, Julie; Zeng, Ruoxi; Choi, Augustine; O'Brien-Ladner, Amy; Dhillon, Navneet K.

    2016-01-01

    ABSTRACT Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids. PMID:27723373

  16. Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension.

    PubMed

    Dalvi, Pranjali; Sharma, Himanshu; Chinnappan, Mahendran; Sanderson, Miles; Allen, Julie; Zeng, Ruoxi; Choi, Augustine; O'Brien-Ladner, Amy; Dhillon, Navneet K

    2016-12-01

    Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.

  17. Failed Regulations and School Composition: Selective Admission Practices in Chilean Primary Schools

    ERIC Educational Resources Information Center

    Carrasco, Alejandro; Gutiérrez, Gabriel; Flores, Carolina

    2017-01-01

    This is an exploratory study about pupil selection. Admission regulations are central to understanding issues of school mix, segregation and educational justice. In Chile, student selection has been intensively discussed but scarcely studied. Using a questionnaire for headteachers (N = 581), we explore how school admission processes are organised…

  18. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

    PubMed Central

    Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

    1989-01-01

    The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted

  19. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

    PubMed Central

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-01-01

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions. PMID:26460481

  20. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

    PubMed

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-10-13

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B-RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

  1. Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

    PubMed

    Sosic, Alice; Saccone, Irene; Carraro, Caterina; Kenderdine, Thomas; Gamba, Elia; Caliendo, Giuseppe; Corvino, Angela; Di Vaio, Paola; Fiorino, Ferdinando; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Severino, Beatrice; Spada, Valentina; Fabris, Dan; Frecentese, Francesco; Gatto, Barbara

    2018-06-12

    The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

  2. A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

    PubMed

    Lin, Min-Hsuan; Sivakumaran, Haran; Jones, Alun; Li, Dongsheng; Harper, Callista; Wei, Ting; Jin, Hongping; Rustanti, Lina; Meunier, Frederic A; Spann, Kirsten; Harrich, David

    2014-12-14

    Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased

  3. HIV-1 Tat-mediated induction of Platelet-derived Growth Factor in Astrocytes: Role of Early Growth Response Gene 1

    PubMed Central

    Bethel-Brown, Crystal; Yao, Honghong; Callen, Shannon; Lee, Young Han; Dash, Prasanta K; Kumar, Anil; Buch, Shilpa

    2011-01-01

    HIV-associated neurological disorders (HAND) are estimated to affect almost 60% of HIV infected individuals. HIV-encephalitis (HIVE), the pathological correlate of the most severe form of HAND is often characterized by glial activation, cytokine/chemokine dysregulation, and neuronal damage and loss. However, the severity of HIVE correlates better with glial activation rather than viral load. Using the macaque model, it has been demonstrated that simian immunodeficiency virus encephalitis (SIVE) correlates with increased expression of the mitogen platelet-derived growth factor-B (PDGF-B) chain in the brain. The present study was aimed at exploring the role of PDGF-B chain in HIV-associated activation and proliferation of astrocytes. Specifically, the data herein demonstrate that exposure of rat and human astrocytes to the HIV-1 protein, Tat resulted in the induction of PDGF at both the mRNA and protein levels. Furthermore, PDGF-BB induction was regulated by activation of ERK1/2 and JNK signaling pathways and the downstream transcription factor, early growth response 1(Egr-1). Chromatin immunoprecipitation (ChIP) assays demonstrated binding of Egr-1 to the PDGF-B promoter. Exposure of astrocytes to PDGF-BB, in turn, led to both increased proliferation and release of pro-inflammatory cytokines MCP-1 and IL-1β. Since astrogliosis is linked to disease severity, understanding its regulation by PDGF-BB could aid in the development of therapeutic intervention strategies for HAND. PMID:21368226

  4. Endocrine regulation and sexual differentiation of avian copulatory sexually selected characters.

    PubMed

    Brennan, Patricia L R; Adkins-Regan, Elizabeth

    2014-10-01

    Reproductive specializations in birds have provided intriguing model systems to better understand the role of endocrine mechanisms that regulate phenotype expression and the action of sexual selection. A comparative approach can elucidate how endocrine systems associated with control of sexual differentiation, sexual maturation, and reproductive physiology and behavior have diversified. Here we compare the copulatory sexually selected traits of two members of the galloanseriform superfamily: quail and ducks. Japanese quail have a non-intromittent penis, and they have evolved a unique foam gland that is known to be involved in post-copulatory sexual selection. In contrast, ducks have maintained a large intromittent penis that has evolved via copulatory male-male competition and has been elaborated in a sexually antagonistic race due to sexual conflict with females over mating. These adaptations function in concert with sex-specific and, in part, species-specific behaviors. Although the approaches to study these traits have been different, exploring the differences in neuroendocrine regulation of sexual behavior, development and seasonality of the foam gland and the penis side by side, allow us to suggest some areas where future research would be productive to better understand the evolution of novelty in sexually selected traits. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Training self-assessment and task-selection skills to foster self-regulated learning: Do trained skills transfer across domains?

    PubMed

    Raaijmakers, Steven F; Baars, Martine; Paas, Fred; van Merriënboer, Jeroen J G; van Gog, Tamara

    2018-01-01

    Students' ability to accurately self-assess their performance and select a suitable subsequent learning task in response is imperative for effective self-regulated learning. Video modeling examples have proven effective for training self-assessment and task-selection skills, and-importantly-such training fostered self-regulated learning outcomes. It is unclear, however, whether trained skills would transfer across domains. We investigated whether skills acquired from training with either a specific, algorithmic task-selection rule or a more general heuristic task-selection rule in biology would transfer to self-regulated learning in math. A manipulation check performed after the training confirmed that both algorithmic and heuristic training improved task-selection skills on the biology problems compared with the control condition. However, we found no evidence that students subsequently applied the acquired skills during self-regulated learning in math. Future research should investigate how to support transfer of task-selection skills across domains.

  6. Training self‐assessment and task‐selection skills to foster self‐regulated learning: Do trained skills transfer across domains?

    PubMed Central

    Baars, Martine; Paas, Fred; van Merriënboer, Jeroen J. G.; van Gog, Tamara

    2018-01-01

    Summary Students' ability to accurately self‐assess their performance and select a suitable subsequent learning task in response is imperative for effective self‐regulated learning. Video modeling examples have proven effective for training self‐assessment and task‐selection skills, and—importantly—such training fostered self‐regulated learning outcomes. It is unclear, however, whether trained skills would transfer across domains. We investigated whether skills acquired from training with either a specific, algorithmic task‐selection rule or a more general heuristic task‐selection rule in biology would transfer to self‐regulated learning in math. A manipulation check performed after the training confirmed that both algorithmic and heuristic training improved task‐selection skills on the biology problems compared with the control condition. However, we found no evidence that students subsequently applied the acquired skills during self‐regulated learning in math. Future research should investigate how to support transfer of task‐selection skills across domains. PMID:29610547

  7. NMR spectroscopic studies of a TAT-derived model peptide in imidazolium-based ILs: influence on chemical shifts and the cis/trans equilibrium state.

    PubMed

    Wiedemann, Christoph; Ohlenschläger, Oliver; Mrestani-Klaus, Carmen; Bordusa, Frank

    2017-09-13

    NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual 1 H and 13 C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.

  8. TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

    PubMed

    Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L

    2017-01-17

    Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

  9. TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

    PubMed Central

    Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

    2017-01-01

    Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

  10. Tat transport of a Sec passenger leads to both completely translocated as well as membrane-arrested passenger proteins.

    PubMed

    Dittmar, Julia; Schlesier, René; Klösgen, Ralf Bernd

    2014-02-01

    We have studied the membrane transport of the chimeric precursor protein 16/33, which is composed of the Tat(1)-specific transport signal of OEC16 and the Sec passenger protein OEC33, both subunits of the oxygen-evolving system associated with photosystem II. Protein transport experiments performed with isolated pea thylakoids show that the 16/33 chimera is transported in a strictly Tat-dependent manner into the thylakoid vesicles yielding mature OEC33 (mOEC33) in two different topologies. One fraction accumulates in the thylakoid lumen and is thus resistant to externally added protease. A second fraction is arrested during transport in an N-in/C-out topology within the membrane. Chase experiments demonstrate that this membrane-arrested mOEC33 moiety does not represent a translocation intermediate but instead an alternative end product of the transport process. Transport arrest of mOEC33, which is embedded in the membrane with a mildly hydrophobic protein segment, requires more than 26 additional and predominantly hydrophilic residues C-terminal of the membrane-embedded segment. Furthermore, it is stimulated by mutations which potentially affect the conformation of mOEC33 suggesting that at least partial folding of the passenger protein is required for complete membrane translocation. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Preparation of TiO2/Ag/TiO2 (TAT) multilayer films with optical and electrical properties enhanced by using Cr-added Ag film

    NASA Astrophysics Data System (ADS)

    Loka, Chadrasekhar; Lee, Kee-Sun

    2017-09-01

    The dielectric-metal-dielectric tri-layer films have attracted much attention by virtue of their low-cost and high quality device performance as a transparent conductive electrode. Here, we report the deposition of Cr doped Ag films sandwiched between thin TiO2 layers and investigation on the surface microstructure, optical and electrical properties depending on the thickness of the Ag(Cr). The activation energy (1.18 eV) for grain growth of Ag was calculated from the Arrhenius plot using the law Dn -D0n = kt , which was comparable to the bulk diffusion of Ag. This result indicated the grain growth of Ag was effectively retarded by the Cr addition, which was presumed to related with blocking the surface and grain boundary diffusion due to Cr segregation. Based on thermal stability of Cr added Ag film, we deposited TiO2/Ag(Cr)/TiO2 (TAT) multilayer thin films and with a 10 nm thick Ag(Cr), the TAT films showed high optical transmittance in the visible region (94.2%), low electrical resistivity (8.66 × 10-5 Ω cm), and hence the high figure of merit 57.15 × 10-3 Ω-1 was achieved. The high transmittance of the TAT film was believed to be attributed to the low optical loss due to a reduction in the Ag layer thickness, the surface plasmon effect, and the electron scattering reduced by the Ag layer with a low electrical resistivity.

  12. 123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1).

    PubMed

    Hu, Meiduo; Chen, Paul; Wang, Judy; Scollard, Deborah A; Vallis, Katherine A; Reilly, Raymond M

    2007-03-01

    To evaluate the internalization and nuclear translocation of (123)I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1). Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21(WAF-1/Cip-1) antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21(WAF-1/Cip-1). Internalization and nuclear translocation were measured. The ability of tat-anti-p21(WAF-1/Cip-1) to block G(1)-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21(WAF-1/Cip-1) was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF. There was 13.4+/-0.2% of radioactivity internalized by MDA-MB-468 cells incubated with (123)I-tat-anti-p21(WAF-1/Cip-1) and 34.6+/-3.1% imported into the nucleus. Tat-anti-p21(WAF-1/Cip-1)(8 muM) decreased the proportion of EGF-treated cells in G(1) phase from 81.9+/-0.7% to 46.1+/-0.7% (p<0.001), almost restoring the G(1) phase fraction to that of unexposed cells (25.8+/-0.2%). Non-specific tat-mouse IgG did not block EGF-induced G(1)-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21(WAF-1/Cip-1) than in mice not receiving EGF (3.1+/-0.4% versus 1.8+/-0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21(WAF-1/Cip-1)/beta-actin ratio. We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible.

  13. Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

    PubMed

    Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

    2006-07-01

    Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

  14. Chronic morphine and HIV-1 Tat promote differential central nervous system trafficking of CD3+ and Ly6C+ immune cells in a murine Streptococcus pneumoniae infection model.

    PubMed

    Dutta, Raini; Roy, Sabita

    2015-06-20

    Persistent systemic infection results in excessive trafficking of peripheral immune cells into the central nervous system (CNS), thereby contributing to sustained neuroinflammation that leads to neurocognitive deficits. In this study, we explored the role of opportunistic systemic infection with Streptococcus pneumoniae in the recruitment of peripheral leukocytes into the CNS and its contribution to HIV-1-associated neurocognitive disorders in opioid-dependent individuals. Wild-type B6CBAF1 (wt), μ-opioid receptor knockout (MORKO), FVB/N luciferase transgenic, and Toll-like receptor 2 and 4 knockout (TLR2KO and TLR4KO) mice were subcutaneously implanted with morphine/placebo pellet followed by HIV-1 Transactivator of transcription (Tat) protein injection intravenously and S. pneumoniae administration intraperitoneally. On postoperative day 5, brains perfused with phosphate-buffered saline were harvested and subjected to immunohistochemistry (for bacterial trafficking and chemokine ligand generation), flow cytometry (for phenotypic characterization of CNS trafficked immune cells), Western blot, and real-time PCR (for ligand expression). Our results show differential leukocyte trafficking of T lymphocytes (CD3+) and inflammatory monocytes (Ly6C+) into the CNS of mice treated with morphine, HIV-1 Tat, and/or S. pneumoniae. In addition, we demonstrate a Trojan horse mechanism for bacterial dissemination across the blood-brain barrier into the CNS by monocytes. Activation of TLRs on microglia induced a chemokine gradient that facilitated receptor-dependent trafficking of peripheral immune cells into the CNS. HIV-1 Tat induced trafficking of Ly6C+ and CD3+ cells into the CNS; infection with S. pneumoniae facilitated infiltration of only T lymphocytes into the CNS. We also observed differential chemokine secretion in the CNS, with CCL5 being the predominant chemokine following HIV-1 Tat treatment, which was potentiated further with morphine. S. pneumoniae alone led to

  15. The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles.

    PubMed

    Smith, Carol-Anne M; de la Fuente, Jesus; Pelaz, Beatriz; Furlani, Edward P; Mullin, Margaret; Berry, Catherine C

    2010-05-01

    Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  16. Mutations in Global Regulators Lead to Metabolic Selection during Adaptation to Complex Environments

    PubMed Central

    Saxer, Gerda; Krepps, Michael D.; Merkley, Eric D.; Ansong, Charles; Deatherage Kaiser, Brooke L.; Valovska, Marie-Thérèse; Ristic, Nikola; Yeh, Ping T.; Prakash, Vittal P.; Leiser, Owen P.; Nakhleh, Luay; Gibbons, Henry S.; Kreuzer, Helen W.; Shamoo, Yousif

    2014-01-01

    Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management

  17. Metal-organic frameworks as selectivity regulators for hydrogenation reactions

    NASA Astrophysics Data System (ADS)

    Zhao, Meiting; Yuan, Kuo; Wang, Yun; Li, Guodong; Guo, Jun; Gu, Lin; Hu, Wenping; Zhao, Huijun; Tang, Zhiyong

    2016-11-01

    Owing to the limited availability of natural sources, the widespread demand of the flavouring, perfume and pharmaceutical industries for unsaturated alcohols is met by producing them from α,β-unsaturated aldehydes, through the selective hydrogenation of the carbon-oxygen group (in preference to the carbon-carbon group). However, developing effective catalysts for this transformation is challenging, because hydrogenation of the carbon-carbon group is thermodynamically favoured. This difficulty is particularly relevant for one major category of heterogeneous catalyst: metal nanoparticles supported on metal oxides. These systems are generally incapable of significantly enhancing the selectivity towards thermodynamically unfavoured reactions, because only the edges of nanoparticles that are in direct contact with the metal-oxide support possess selective catalytic properties; most of the exposed nanoparticle surfaces do not. This has inspired the use of metal-organic frameworks (MOFs) to encapsulate metal nanoparticles within their layers or inside their channels, to influence the activity of the entire nanoparticle surface while maintaining efficient reactant and product transport owing to the porous nature of the material. Here we show that MOFs can also serve as effective selectivity regulators for the hydrogenation of α,β-unsaturated aldehydes. Sandwiching platinum nanoparticles between an inner core and an outer shell composed of an MOF with metal nodes of Fe3+, Cr3+ or both (known as MIL-101; refs 19, 20, 21) results in stable catalysts that convert a range of α,β-unsaturated aldehydes with high efficiency and with significantly enhanced selectivity towards unsaturated alcohols. Calculations reveal that preferential interaction of MOF metal sites with the carbon-oxygen rather than the carbon-carbon group renders hydrogenation of the former by the embedded platinum nanoparticles a thermodynamically favoured reaction. We anticipate that our basic design

  18. Metal-organic frameworks as selectivity regulators for hydrogenation reactions.

    PubMed

    Zhao, Meiting; Yuan, Kuo; Wang, Yun; Li, Guodong; Guo, Jun; Gu, Lin; Hu, Wenping; Zhao, Huijun; Tang, Zhiyong

    2016-11-03

    Owing to the limited availability of natural sources, the widespread demand of the flavouring, perfume and pharmaceutical industries for unsaturated alcohols is met by producing them from α,β-unsaturated aldehydes, through the selective hydrogenation of the carbon-oxygen group (in preference to the carbon-carbon group). However, developing effective catalysts for this transformation is challenging, because hydrogenation of the carbon-carbon group is thermodynamically favoured. This difficulty is particularly relevant for one major category of heterogeneous catalyst: metal nanoparticles supported on metal oxides. These systems are generally incapable of significantly enhancing the selectivity towards thermodynamically unfavoured reactions, because only the edges of nanoparticles that are in direct contact with the metal-oxide support possess selective catalytic properties; most of the exposed nanoparticle surfaces do not. This has inspired the use of metal-organic frameworks (MOFs) to encapsulate metal nanoparticles within their layers or inside their channels, to influence the activity of the entire nanoparticle surface while maintaining efficient reactant and product transport owing to the porous nature of the material. Here we show that MOFs can also serve as effective selectivity regulators for the hydrogenation of α,β-unsaturated aldehydes. Sandwiching platinum nanoparticles between an inner core and an outer shell composed of an MOF with metal nodes of Fe 3+ , Cr 3+ or both (known as MIL-101; refs 19, 20, 21) results in stable catalysts that convert a range of α,β-unsaturated aldehydes with high efficiency and with significantly enhanced selectivity towards unsaturated alcohols. Calculations reveal that preferential interaction of MOF metal sites with the carbon-oxygen rather than the carbon-carbon group renders hydrogenation of the former by the embedded platinum nanoparticles a thermodynamically favoured reaction. We anticipate that our basic design

  19. Use of the TAT in the assessment of DSM-IV cluster B personality disorders.

    PubMed

    Ackerman, S J; Clemence, A J; Weatherill, R; Hilsenroth, M J

    1999-12-01

    The Social Cognition and Object Relations Scale (SCORS), developed by Western, Lohr, Silk, Kerber, and Goodrich (1985), is a diagnostic instrument used to assess an array of psychological functioning by using clinical narratives such as the Thematic Apperception Test (TAT; Murray, 1943) stories. This study investigated the utility of the SCORS to differentiate between Diagnostic and Statistical Manual of Mental Disorders (4th ed. [DSM-IV]; American Psychiatric Association, 1994) antisocial personality disorder (ANPD), borderline personality disorder (BPD), narcissistic personality disorder (NPD), and Cluster C personality disorder (CPD). A sample of 58 patients was separated into four groups: ANPD (n = 9), BPD (n = 21; 18 with a primary BPD diagnosis and 3 with prominent borderline traits who met 4 of the 5 DSM-IV criteria necessary for a BPD diagnosis), NPD (n = 16; 8 with a primary NPD diagnosis and 8 with prominent narcissistic traits who met 4 of the 5 DSM-IV criteria necessary for a NPD diagnosis), and CPD (n = 12). These groups were then compared on the 8 SCORS variables by using 5 TAT cards (1, 2, 3BM, 4, and 13MF). Spearman-Brown correction for 2-way mixed effects model of reliability for the 8 SCORS variables ranged from .70 to .95. The results of categorical and dimensional analyses indicate that (a) SCORS variables can be used to differentiate ANPD, BPD, and NPD; (b) the BPD group scored significantly lower (greater maladjustment) than did the CPD group on certain variables; (c) the BPD group scored significantly lower (greater maladjustment) than did the NPD group on all 8 SCORS variables; (d) the ANPD group scored significantly lower than did the NPD group on certain variables; (e) certain variables were found to be empirically related to the total number of DSM-IV ANPD, BPD, and NPD criteria; and (f) certain variables were found to be empirically related to Minnesota Multiphasic Personality Inventory-2 (MMPI-2; Butcher, Dahlstrom, Graham, Tellegen

  20. Emotion regulation strategy selection in daily life: The role of social context and goals.

    PubMed

    English, Tammy; Lee, Ihno A; John, Oliver P; Gross, James J

    2017-04-01

    Recent studies have begun to document the diversity of ways people regulate their emotions. However, one unanswered question is why people regulate their emotions as they do in everyday life. In the present research, we examined how social context and goals influence strategy selection in daily high points and low points. As expected, suppression was particularly tied to social features of context: it was used more when others were present, especially non-close partners, and when people had instrumental goals, especially more interpersonal ones (e.g., avoid conflict). Distraction and reappraisal were used more when regulating for hedonic reasons (e.g., to feel better), but these strategies were also linked to certain instrumental goals (e.g., getting work done). When contra-hedonic regulation occurred, it primarily took the form of dampening positive emotion during high points. Suppression was more likely to be used for contra-hedonic regulation, whereas reappraisal and distraction were used more for pro-hedonic regulation. Overall, these findings highlight the social nature of emotion regulation and underscore the importance of examining regulation in both positive and negative contexts.

  1. Emotion regulation strategy selection in daily life: The role of social context and goals

    PubMed Central

    Lee, Ihno A.; John, Oliver P.; Gross, James J.

    2016-01-01

    Recent studies have begun to document the diversity of ways people regulate their emotions. However, one unanswered question is why people regulate their emotions as they do in everyday life. In the present research, we examined how social context and goals influence strategy selection in daily high points and low points. As expected, suppression was particularly tied to social features of context: it was used more when others were present, especially non-close partners, and when people had instrumental goals, especially more interpersonal ones (e.g., avoid conflict). Distraction and reappraisal were used more when regulating for hedonic reasons (e.g., to feel better), but these strategies were also linked to certain instrumental goals (e.g., getting work done). When contra-hedonic regulation occurred, it primarily took the form of dampening positive emotion during high points. Suppression was more likely to be used for contra-hedonic regulation, whereas reappraisal and distraction were used more for pro-hedonic regulation. Overall, these findings highlight the social nature of emotion regulation and underscore the importance of examining regulation in both positive and negative contexts. PMID:28652647

  2. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

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

    Jung, Kyung oh; Biomedical Sciences, Seoul National University College of Medicine; Cancer Research Institute, Seoul National University College of Medicine

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescencemore » signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.« less

  3. Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein.

    PubMed

    Kulkarni, Archana; Kurle, Swarali; Shete, Ashwini; Ghate, Manisha; Godbole, Sheela; Madhavi, Vijaya; Kent, Stephen J; Paranjape, Ramesh; Thakar, Madhuri

    2017-01-01

    HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm 3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm 3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288-330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection.

  4. Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

    PubMed

    Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C; Fiorini, Claudia; Ludwig, Leif S; McFarland, Sean K; Abdulhay, Nour J; Specht, Harrison; Keshishian, Hasmik; Mani, D R; Jovanovic, Marko; Ellis, Steven R; Fulco, Charles P; Engreitz, Jesse M; Schütz, Sabina; Lian, John; Gripp, Karen W; Weinberg, Olga K; Pinkus, Geraldine S; Gehrke, Lee; Regev, Aviv; Lander, Eric S; Gazda, Hanna T; Lee, Winston Y; Panse, Vikram G; Carr, Steven A; Sankaran, Vijay G

    2018-03-22

    Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Lack of miR-133a Decreases Contractility of Diabetic Hearts: A Role for Novel Cross Talk Between Tyrosine Aminotransferase and Tyrosine Hydroxylase

    PubMed Central

    Nandi, Shyam Sundar; Zheng, Hong; Sharma, Neeru M.; Shahshahan, Hamid R.; Patel, Kaushik P.

    2016-01-01

    MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat’s heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function. PMID:27411382

  6. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones.

    PubMed

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-06-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

  7. 45 CFR 660.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 3 2012-10-01 2012-10-01 false What procedures apply to the selection of programs and activities under these regulations? 660.6 Section 660.6 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL SCIENCE FOUNDATION INTERGOVERNMENTAL REVIEW OF THE NATIONAL SCIENCE...

  8. 45 CFR 660.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 3 2013-10-01 2013-10-01 false What procedures apply to the selection of programs and activities under these regulations? 660.6 Section 660.6 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL SCIENCE FOUNDATION INTERGOVERNMENTAL REVIEW OF THE NATIONAL SCIENCE...

  9. 45 CFR 660.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 3 2011-10-01 2011-10-01 false What procedures apply to the selection of programs and activities under these regulations? 660.6 Section 660.6 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL SCIENCE FOUNDATION INTERGOVERNMENTAL REVIEW OF THE NATIONAL SCIENCE...

  10. 45 CFR 660.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 3 2014-10-01 2014-10-01 false What procedures apply to the selection of programs and activities under these regulations? 660.6 Section 660.6 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL SCIENCE FOUNDATION INTERGOVERNMENTAL REVIEW OF THE NATIONAL SCIENCE...

  11. 45 CFR 660.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false What procedures apply to the selection of programs and activities under these regulations? 660.6 Section 660.6 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL SCIENCE FOUNDATION INTERGOVERNMENTAL REVIEW OF THE NATIONAL SCIENCE...

  12. RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration.

    PubMed

    Wu, Dongni; Zhang, Yongnu; Xu, Xiaoting; Guo, Ting; Xie, Deming; Zhu, Rong; Chen, Shengfeng; Ramakrishna, Seeram; He, Liumin

    2018-05-01

    In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1 H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. We successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex

  13. Mobile Eye Tracking Reveals Little Evidence for Age Differences in Attentional Selection for Mood Regulation

    PubMed Central

    Isaacowitz, Derek M.; Livingstone, Kimberly M.; Harris, Julia A.; Marcotte, Stacy L.

    2014-01-01

    We report two studies representing the first use of mobile eye tracking to study emotion regulation across adulthood. Past research on age differences in attentional deployment using stationary eye tracking has found older adults show relatively more positive looking, and seem to benefit more mood-wise from this looking pattern, compared to younger adults. However, these past studies have greatly constrained the stimuli participants can look at, despite real-world settings providing numerous possibilities for what to choose to look at. We therefore used mobile eye tracking to study age differences in attentional selection, as indicated by fixation patterns to stimuli of different valence freely chosen by the participant. In contrast to stationary eye tracking studies of attentional deployment, Study 1 showed that younger and older individuals generally selected similar proportions of valenced stimuli, and attentional selection had similar effects on mood across age groups. Study 2 replicated this pattern with an adult lifespan sample including middle-aged individuals. Emotion regulation-relevant attention may thus differ depending on whether stimuli are freely chosen or not. PMID:25527965

  14. Recent advances in the identification of Tat-mediated transactivation inhibitors: progressing toward a functional cure of HIV.

    PubMed

    Tabarrini, Oriana; Desantis, Jenny; Massari, Serena

    2016-01-01

    The current anti-HIV combination therapy does not eradicate the virus that persists mainly in quiescent infected CD4(+) T cells as a latent integrated provirus that resumes after therapy interruption. The Tat-mediated transactivation (TMT) is a critical step in the HIV replication cycle that could give the opportunity to reduce the size of latent reservoirs. More than two decades of research led to the identification of various TMT inhibitors. While none of them met the criteria to reach the market, the search for a suitable TMT inhibitor is still actively pursued. Really promising compounds, including one in a Phase III clinical trial, have been recently identified, thus warranting an update.

  15. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

    PubMed Central

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-01-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets. PMID:23605042

  16. PDRG1, a novel tumor marker for multiple malignancies that is selectively regulated by genotoxic stress

    PubMed Central

    Jiang, Lingyan; Luo, Xiuquan; Shi, Jingxue; Sun, Hong; Sun, Qing; Sheikh, M Saeed

    2011-01-01

    We have previously cloned and characterized a novel p53 and DNA damage-regulated gene named PDRG1. PDRG1 was found to be differentially regulated by ultraviolet (UV) radiation and p53. In this study, we further investigated stress regulation of PDRG1 and found it to be selectively regulated by agents that induce genotoxic stress (DNA damage). Using cancer profiling arrays, we also investigated PDRG1 expression in matching normal and tumor samples representing various malignancies and found its expression to be upregulated in multiple malignancies including cancers of the colon, rectum, ovary, lung, stomach, breast and uterus when compared to their respective matched normal tissues. Western blot and immunohistochemical analyses were also performed on select specimen sets of colon cancers and matching normal tissues and the results also indicated PDRG1 overexpression in tumors relative to normal tissues. To gain insight into the function of PDRG1, we performed PDRG1 knockdown in human colon cancer cells and found its depletion to result in marked slowdown of tumor cell growth. These results suggest that PDRG1 may be linked to cell growth regulation. Yeast two-hybrid screening also led to the identification of PDCD7, CIZ1 and MAP1S as PDRG1-interacting proteins that are involved in apoptosis and cell cycle regulation which further implicate PDRG1 in controlling cell growth regulation. Taken together, our results indicate that PDRG1 expression is increased in multiple human malignancies suggesting it to be a high-value novel tumor marker that could play a role in cancer development and/or progression. PMID:21193842

  17. Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

    PubMed

    Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

    1995-07-04

    Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells.

  18. 43 CFR 9.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Secretary of changes in their program selections. (d) The Secretary uses a state's process as soon as... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false What procedures apply to the selection of programs and activities under these regulations? 9.6 Section 9.6 Public Lands: Interior Office of the...

  19. Training Self-Regulated Learning Skills with Video Modeling Examples: Do Task-Selection Skills Transfer?

    ERIC Educational Resources Information Center

    Raaijmakers, Steven F.; Baars, Martine; Schaap, Lydia; Paas, Fred; van Merriënboer, Jeroen; van Gog, Tamara

    2018-01-01

    Self-assessment and task-selection skills are crucial in self-regulated learning situations in which students can choose their own tasks. Prior research suggested that training with video modeling examples, in which another person (the model) demonstrates and explains the cyclical process of problem-solving task performance, self-assessment, and…

  20. You mob my owl, I'll mob yours: birds play tit-for-tat game.

    PubMed

    Krama, Tatjana; Vrublevska, Jolanta; Freeberg, Todd M; Kullberg, Cecilia; Rantala, Markus J; Krams, Indrikis

    2012-01-01

    Reciprocity is fundamental to cooperative behaviour and has been verified in theoretical models. However, there is still limited experimental evidence for reciprocity in non-primate species. Our results more decisively clarify that reciprocity with a tit-for-tat enforcement strategy can occur among breeding pied flycatchers Ficedula hypoleuca separate from considerations of byproduct mutualism. Breeding pairs living in close proximity (20-24 m) did exhibit byproduct mutualism and always assisted in mobbing regardless of their neighbours' prior actions. However, breeding pairs with distant neighbours (69-84 m) either assisted or refused to assist in mobbing a predatory owl based on whether or not the distant pair had previously helped them in their own nest defense against the predator. Clearly, these birds are aware of their specific spatial security context, remember their neighbours' prior behaviour, and choose a situation-specific strategic course of action, which could promote their longer-term security, a capacity previously thought unique to primates.

  1. You mob my owl, I'll mob yours: birds play tit-for-tat game

    PubMed Central

    Krama, Tatjana; Vrublevska, Jolanta; Freeberg, Todd M.; Kullberg, Cecilia; Rantala, Markus J.; Krams, Indrikis

    2012-01-01

    Reciprocity is fundamental to cooperative behaviour and has been verified in theoretical models. However, there is still limited experimental evidence for reciprocity in non-primate species. Our results more decisively clarify that reciprocity with a tit-for-tat enforcement strategy can occur among breeding pied flycatchers Ficedula hypoleuca separate from considerations of byproduct mutualism. Breeding pairs living in close proximity (20–24 m) did exhibit byproduct mutualism and always assisted in mobbing regardless of their neighbours' prior actions. However, breeding pairs with distant neighbours (69–84 m) either assisted or refused to assist in mobbing a predatory owl based on whether or not the distant pair had previously helped them in their own nest defense against the predator. Clearly, these birds are aware of their specific spatial security context, remember their neighbours' prior behaviour, and choose a situation-specific strategic course of action, which could promote their longer-term security, a capacity previously thought unique to primates. PMID:23150772

  2. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex

    PubMed Central

    Demarsy, Emilie; Lakshmanan, Ashok M.; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC–TIC (translocon of outer membrane complex–translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms. PMID:25278954

  3. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

    PubMed

    Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

  4. Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

    PubMed

    Chichger, Havovi; Braza, Julie; Duong, Huetran; Boni, Geraldine; Harrington, Elizabeth O

    2016-06-01

    Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

  5. Central leptin regulates heart lipid content by selectively increasing PPAR β/δ expression.

    PubMed

    Mora, Cristina; Pintado, Cristina; Rubio, Blanca; Mazuecos, Lorena; López, Virginia; Fernández, Alejandro; Salamanca, Aurora; Bárcena, Brenda; Fernández-Agulló, Teresa; Arribas, Carmen; Gallardo, Nilda; Andrés, Antonio

    2018-01-01

    The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d ) and their target genes, adipose triglyceride lipase (encoded by Pnpla2 , herefater referred to as Atgl ), hormone sensitive lipase (encoded by Lipe , herefater referred to as Hsl ), pyruvate dehydrogenase kinase 4 ( Pdk4 ) and acyl CoA oxidase 1 ( Acox1 ), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 ( Scd1 ) and diacylglycerol acyltransferase 1 ( Dgat1 ) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight. © 2018 Society for Endocrinology.

  6. Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release.

    PubMed

    Dupuis, Amandine; Wattiez, Anne-Sophie; Pinguet, Jérémy; Richard, Damien; Libert, Frédéric; Chalus, Maryse; Aissouni, Youssef; Sion, Benoit; Ardid, Denis; Marin, Philippe; Eschalier, Alain; Courteix, Christine

    2017-04-01

    Antidepressants are one of the first line treatments for neuropathic pain but their use is limited by the incidence and severity of side effects of tricyclics and the weak effectiveness of selective serotonin reuptake inhibitors (SSRIs). Serotonin type 2A (5-HT 2A ) receptors interact with PDZ proteins that regulate their functionality and SSRI efficacy to alleviate pain. We investigated whether an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT 2A receptors and associated PDZ proteins would improve the treatment of traumatic neuropathic allodynia. Tactile allodynia was assessed in spinal nerve ligation-induced neuropathic pain in rats using von Frey filaments after acute treatment with TAT-2ASCV and/or 5-HT 2A receptor agonist, alone or in combination with repeated treatment with fluoxetine. In vivo microdialysis was performed in order to examine the involvement of GABA in TAT-2ASCV/fluoxetine treatment-associated analgesia. TAT-2ASCV (100ng, single i.t. injection) improved SNL-induced tactile allodynia by increasing 5-HT 2A receptor responsiveness to endogenous 5-HT. Fluoxetine alone (10mg/kg, five i.p. injections) slightly increased tactile thresholds and its co-administration with TAT-2ASCV (100ng, single i.t. injection) further enhanced the anti-allodynic effect. This effect depends on the integrity of descending serotonergic bulbospinal pathways and spinal release of GABA. The anti-allodynic effect of fluoxetine can be enhanced by disrupting 5-HT 2A receptor-PDZ protein interactions. This enhancement depends on 5-HT 2A receptor activation, spinal GABA release and GABAA receptor activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Directional selectivity of afferent neurons in zebrafish neuromasts is regulated by Emx2 in presynaptic hair cells

    PubMed Central

    Ji, Young Rae; Warrier, Sunita; Jiang, Tao

    2018-01-01

    The orientation of hair bundles on top of sensory hair cells (HCs) in neuromasts of the lateral line system allows fish to detect direction of water flow. Each neuromast shows hair bundles arranged in two opposing directions and each afferent neuron innervates only HCs of the same orientation. Previously, we showed that this opposition is established by expression of Emx2 in half of the HCs, where it mediates hair bundle reversal (Jiang et al., 2017). Here, we show that Emx2 also regulates neuronal selection: afferent neurons innervate either Emx2-positive or negative HCs. In emx2 knockout and gain-of-function neuromasts, all HCs are unidirectional and the innervation patterns and physiological responses of the afferent neurons are dependent on the presence or absence of Emx2. Our results indicate that Emx2 mediates the directional selectivity of neuromasts by two distinct processes: regulating hair bundle orientation in HCs and selecting afferent neuronal targets. PMID:29671737

  8. Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin.

    PubMed

    Khamehchian, Sedigheh; Nikkhah, Maryam; Madani, Rasool; Hosseinkhani, Saman

    2016-11-01

    Functionalization of gold nanoparticles (GNPs) is suitable for many applications such as biomedical imaging, clinical diagnosis, and targeted delivery by conjugating cell-penetrating peptides (CPPs). Here, we investigated intracellular uptake of GNP conjugated to MCaUF1-9(Ala) , a CPP derived from maurocalcine (MCa) animal toxin, and compared it with TAT functionalized GNP. Peptide conjugated GNP was characterized using UV-Visible spectroscopy, dynamic light scattering, zeta potential, and transmission electron microscopy. Uptake of MCaUF1-9(Ala) and TAT functionalized GNPs was evaluated in three cell lines, HeLa, MDA-MB-231, and A431, using dark field imaging and atomic absorption spectroscopy. According to peptide sequences and type of cells different cell penetrating activity was observed. Peptide functionalized GNP had little effect on cell viability and respect to net charge difference between peptide, showed interesting selectivity against three cell types. Peptide conjugated to GNPs displayed higher uptake than bare GNPs in the all cell lines except HeLa cell with lowest internalization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2693-2700, 2016. © 2016 Wiley Periodicals, Inc.

  9. Mutations in global regulators lead to metabolic selection during adaptation to complex environments

    DOE PAGES

    Saxer, Gerda; Krepps, Michael D.; Merkley, Eric D.; ...

    2014-12-11

    Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Unlike adaptation to a single limiting resource, adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes since many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased geneticmore » and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that a subtle modulation of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order “metabolic selection” that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel

  10. α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

    PubMed Central

    Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

    2016-01-01

    Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

  11. Effects of the Ordering of Natural Selection and Population Regulation Mechanisms on Wright-Fisher Models

    PubMed Central

    He, Zhangyi; Beaumont, Mark; Yu, Feng

    2017-01-01

    We explore the effect of different mechanisms of natural selection on the evolution of populations for one- and two-locus systems. We compare the effect of viability and fecundity selection in the context of the Wright-Fisher model with selection under the assumption of multiplicative fitness. We show that these two modes of natural selection correspond to different orderings of the processes of population regulation and natural selection in the Wright-Fisher model. We find that under the Wright-Fisher model these two different orderings can affect the distribution of trajectories of haplotype frequencies evolving with genetic recombination. However, the difference in the distribution of trajectories is only appreciable when the population is in significant linkage disequilibrium. We find that as linkage disequilibrium decays the trajectories for the two different models rapidly become indistinguishable. We discuss the significance of these findings in terms of biological examples of viability and fecundity selection, and speculate that the effect may be significant when factors such as gene migration maintain a degree of linkage disequilibrium. PMID:28500051

  12. Epigenetic regulation of the oxytocin receptor is associated with neural response during selective social attention.

    PubMed

    Puglia, Meghan H; Connelly, Jessica J; Morris, James P

    2018-06-15

    Aberrant attentional biases to social stimuli have been implicated in a number of disorders including autism and social anxiety disorder. Oxytocin, a naturally-occurring mammalian hormone and neuromodulator involved in regulating social behavior, has been proposed to impact basic biological systems that facilitate the detection of and orientation to social information. Here, we investigate a role for naturally-occurring variability in the endogenous oxytocinergic system in regulating neural response during attention to social information. Participants performed a selective social attention task while undergoing fMRI, provided a blood sample for epigenetic analysis, and completed self-report measures of social functioning. We find that a functional epigenetic modification to the oxytocin receptor, OXTR methylation, is associated with increased neural response within and decreased functional coupling between regions of the salience and attentional control networks during selective social attention. We also show that subclinical variability in autistic and social anxiety traits moderates this epigenetic regulation of neural response. These data offer a mechanistic explanation to a growing literature associating social behavior and disorder with epigenetic modification to OXTR by suggesting that OXTR methylation reflects a decrease in the extent to which social information automatically captures attention. We highlight the importance that treatment efficacy be considered in relation to individual differences in molecular makeup, and that future studies aimed at uncovering biomarkers of disorder carefully consider measurement at both the biological and phenotypic level.

  13. Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

    PubMed

    Hu, Zhenyu; Li, Guang; Wang, Jiong-Wei; Chong, Suet Yen; Yu, Dejie; Wang, Xiaoyuan; Soon, Jia Lin; Liang, Mui Cheng; Wong, Yuk Peng; Huang, Na; Colecraft, Henry M; Liao, Ping; Soong, Tuck Wah

    2018-04-12

    Background -L-type Ca V 1.2 channels play crucial roles in regulation of blood pressure. Galectin-1 (Gal-1), has been reported to bind to the I-II loop of Ca V 1.2 channels to reduce their current density. However, the mechanistic understanding for the down-regulation of Ca V 1.2 channels by Gal-1, and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. Methods - In vitro experiments involving co-IP, western blot, patch-clamp recordings, immunohistochemistry and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 down-regulates Ca V 1.2 channel in transfected HEK 293 cells, smooth muscle cells, arteries from Lgasl1 -/- mice, rat and human patients. In vivo experiments involving delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting Ca V 1.2-Gal-1 interaction on blood pressure monitored by tail cuff or telemetry methods. Results -Our study reveals that Gal-1 is a key regulator for proteasomal degradation of Ca V 1.2 channels. Gal-1 competed allosterically with Ca V β subunit for binding to the I-II loop of Ca V 1.2 channel. This competitive disruption of Ca V β binding led to Ca V 1.2 degradation by exposing the channels to poly-ubiquitination. Notably, we demonstrated that the inverse relationship of reduced Gal-1 and increased Ca V 1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice due to up-regulated Ca V 1.2 protein level in arteries. To directly regulate blood pressure by targeting the Ca V 1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1, by a mini-osmotic pump and this specific disruption of Ca V 1.2-Gal-1 coupling increased smooth muscle Ca V 1.2 currents, induced larger arterial contraction and caused hypertension in rats. In contrasting experiments, over-expression of Gal-1 in smooth muscle by a

  14. Select Agent and Toxin Regulations: Beyond the Eighth Edition of the Guide for the Care and Use of Laboratory Animals

    PubMed Central

    Kastenmayer, Robin J; Moore, Rashida M; Bright, Allison L; Torres-Cruz, Rafael; Elkins, William R

    2012-01-01

    In the interval between the publication of the seventh and eighth editions of the Guide for the Care and Use of Laboratory Animals (Guide), much has changed with regard to the regulation and funding of highly pathogenic biologic agents and toxins (Select Agents). Funding of research involving highly pathogenic agents has increased dramatically during this time, thus increasing the demand for facilities capable of supporting this work. The eighth edition of the Guide briefly mentions Select Agents and provides a limited set of references. Here we provide some background information regarding the relevant laws and regulations, as well as an overview of the programmatic requirements pertaining to the use of Select Agents, with a focus on use in animals. PMID:22776191

  15. Involvement of Phosphatidylinositol 3-Kinase-Mediated Up-Regulation of IκBα in Anti-Inflammatory Effect of Gemfibrozil in Microglia1

    PubMed Central

    Jana, Malabendu; Jana, Arundhati; Liu, Xiaojuan; Ghosh, Sankar; Pahan, Kalipada

    2008-01-01

    The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-α (PPAR-α) in microglial cells and isolating primary microglia from PPAR-α−/− mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-α. Interestingly, gemfibrozil induced the activation of p85α-associated PI3K (p110β but not p110α) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid β (Aβ)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-γ-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Aβ-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-κB activation in LPS-, Aβ-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-γ-, induced microglial expression of iNOS and stimulation of IκBα expression and inhibition of NF-κB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-κB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IκBα. PMID:17785853

  16. Childhood football play and practice in relation to self-regulation and national team selection; a study of Norwegian elite youth players.

    PubMed

    Erikstad, Martin K; Høigaard, Rune; Johansen, Bjørn Tore; Kandala, Ngianga-Bakwin; Haugen, Tommy

    2018-03-09

    Childhood sport participation is argued to be important to understand differences in self-regulation and performance level in adolescence. This study sought to investigate if football-specific activities in childhood (6-12 years of age) is related to self-regulatory skills and national under 14- and 15-team selection in Norwegian elite youth football. Data of practice histories and self-regulatory skills of 515 youth football players selected at Norwegian regional level were collected and further analysed using multilevel analyses. The results revealed that high self-regulated players were more likely to be selected for national initiatives, and increased their involvement in peer-led football practice and adult-led football practice during childhood, compared to players with lower levels of self-regulation. While national level players reported higher levels of peer-led football play in childhood, the interaction effect suggest that the regional level players increased their involvement in peer-led play during childhood compared to national level players. In conclusion, the findings indicate that childhood sport participation may contribute to later differences in self-regulation, and highlights the importance of childhood engagement in football-specific play and practice in the development of Norwegian youth football players.

  17. A quantitative genetic model of reciprocal altruism: a condition for kin or group selection to prevail.

    PubMed Central

    Aoki, K

    1983-01-01

    A condition is derived for reciprocal altruism to evolve by kin or group selection. It is assumed that many additively acting genes of small effect and the environment determine the probability that an individual is a reciprocal altruist, as opposed to being unconditionally selfish. The particular form of reciprocal altruism considered is TIT FOR TAT, a strategy that involves being altruistic on the first encounter with another individual and doing whatever the other did on the previous encounter in subsequent encounters with the same individual. Encounters are restricted to individuals of the same generation belonging to the same kin or breeding group, but first encounters occur at random within that group. The number of individuals with which an individual interacts is assumed to be the same within any kin or breeding group. There are 1 + i expected encounters between two interacting individuals. On any encounter, it is assumed that an individual who behaves altruistically suffers a cost in personal fitness proportional to c while improving his partner's fitness by the same proportion of b. Then, the condition for kin or group selection to prevail is [Formula: see text] if group size is sufficiently large and the group mean and the within-group genotypic variance of the trait value (i.e., the probability of being a TIT-FOR-TAT strategist) are uncorrelated. Here, C, Vb, and Tb are the population mean, between-group variance, and between-group third central moment of the trait value and r is the correlation between the additive genotypic values of interacting kin or of individuals within the same breeding group. The right-hand side of the above inequality is monotone decreasing in C if we hold Tb/Vb constant, and kin and group selection become superfluous beyond a certain threshold value of C. The effect of finite group size is also considered in a kin-selection model. PMID:6575395

  18. The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

    PubMed

    Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

    2016-10-14

    Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

    PubMed Central

    BRADRICK, THOMAS D.; MARINO, JOHN P.

    2004-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (Δ TAR-ap23 and Δ TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP ΔTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD ~ 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD ~ 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the ΔTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD ~ 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD ~ 4 μM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions. PMID:15273324

  20. Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough

    PubMed Central

    Mazzone, Stuart B.; Reynolds, Sandra M.; Mori, Nanako; Kollarik, Marian; Farmer, David G.; Myers, Allen C.

    2009-01-01

    We have identified a distinct subtype of airway vagal afferent nerve that plays an essential role in regulating the cough reflex. These afferents are exquisitely sensitive to punctate mechanical stimuli, acid, and decreases in extracellular chloride concentrations, but are insensitive to capsaicin, bradykinin, histamine, adenosine, serotonin, or changes in airway intraluminal pressures. In this study we used intravital imaging, retrograde neuronal tracing, and electrophysiological analyses to characterize the structural basis for their peculiar mechanical sensitivity and to further characterize the regulation of their excitability. In completing these experiments, we uncovered evidence for an essential role of an isozyme of Na+-K+ ATPase in regulating cough. These vagal sensory neurons arise bilaterally from the nodose ganglia and are selectively and brilliantly stained intravitally with the styryl dye FM2-10. Cough receptor terminations are confined and adherent to the extracellular matrix separating the airway epithelium and smooth muscle layers, a site of extensive remodeling in asthma and chronic obstructive pulmonary disease. The cough receptor terminals uniquely express the α3 subunit of Na+-K+ ATPase. Intravital staining of cough receptors by FM2-10, cough receptor excitability in vitro, and coughing in vivo are potently and selectively inhibited by the sodium pump inhibitor ouabain. These data provide the first detailed morphological description of the peripheral terminals of the sensory nerves regulating cough and identify a selective molecular target for their modulation. PMID:19864578

  1. Regional selection of the brain size regulating gene CASC5 provides new insight into human brain evolution.

    PubMed

    Shi, Lei; Hu, Enzhi; Wang, Zhenbo; Liu, Jiewei; Li, Jin; Li, Ming; Chen, Hua; Yu, Chunshui; Jiang, Tianzi; Su, Bing

    2017-02-01

    Human evolution is marked by a continued enlargement of the brain. Previous studies on human brain evolution focused on identifying sequence divergences of brain size regulating genes between humans and nonhuman primates. However, the evolutionary pattern of the brain size regulating genes during recent human evolution is largely unknown. We conducted a comprehensive analysis of the brain size regulating gene CASC5 and found that in recent human evolution, CASC5 has accumulated many modern human specific amino acid changes, including two fixed changes and six polymorphic changes. Among human populations, 4 of the 6 amino acid polymorphic sites have high frequencies of derived alleles in East Asians, but are rare in Europeans and Africans. We proved that this between-population allelic divergence was caused by regional Darwinian positive selection in East Asians. Further analysis of brain image data of Han Chinese showed significant associations of the amino acid polymorphic sites with gray matter volume. Hence, CASC5 may contribute to the morphological and structural changes of the human brain during recent evolution. The observed between-population divergence of CASC5 variants was driven by natural selection that tends to favor a larger gray matter volume in East Asians.

  2. Using generalized linear models to estimate selectivity from short-term recoveries of tagged red drum Sciaenops ocellatus: Effects of gear, fate, and regulation period

    USGS Publications Warehouse

    Burdick, Summer M.; Hightower, Joseph E.; Bacheler, Nathan M.; Paramore, Lee M.; Buckel, Jeffrey A.; Pollock, Kenneth H.

    2010-01-01

    Estimating the selectivity patterns of various fishing gears is a critical component of fisheries stock assessment due to the difficulty in obtaining representative samples from most gears. We used short-term recoveries (n = 3587) of tagged red drum Sciaenops ocellatus to directly estimate age- and length-based selectivity patterns using generalized linear models. The most parsimonious models were selected using AIC, and standard deviations were estimated using simulations. Selectivity of red drum was dependent upon the regulation period in which the fish was caught, the gear used to catch the fish (i.e., hook-and-line, gill nets, pound nets), and the fate of the fish upon recovery (i.e., harvested or released); models including all first-order interactions between main effects outperformed models without interactions. Selectivity of harvested fish was generally dome-shaped and shifted toward larger, older fish in response to regulation changes. Selectivity of caught-and-released red drum was highest on the youngest and smallest fish in the early and middle regulation periods, but increased on larger, legal-sized fish in the late regulation period. These results suggest that catch-and-release mortality has consistently been high for small, young red drum, but has recently become more common in larger, older fish. This method of estimating selectivity from short-term tag recoveries is valuable because it is simpler than full tag-return models, and may be more robust because yearly fishing and natural mortality rates do not need to be modeled and estimated.

  3. Regulation of Histone Acetyltransferase TIP60 Function by Histone Deacetylase 3

    PubMed Central

    Yi, Jingjie; Huang, Xiangyang; Yang, Yuxia; Zhu, Wei-Guo; Gu, Wei; Luo, Jianyuan

    2014-01-01

    The key member of the MOZ (monocyticleukaemia zinc finger protein), Ybf2/Sas3, Sas2, and TIP60 acetyltransferases family, Tat-interactive protein, 60 kD (TIP60), tightly modulates a wide array of cellular processes, including chromatin remodeling, gene transcription, apoptosis, DNA repair, and cell cycle arrest. The function of TIP60 can be regulated by SIRT1 through deacetylation. Here we found that TIP60 can also be functionally regulated by HDAC3. We identified six lysine residues as its autoacetylation sites. Mutagenesis of these lysines to arginines completely abolishes the autoacetylation of TIP60. Overexpression of HDAC3 increases TIP60 ubiquitination levels. However, unlike SIRT1, HDAC3 increased the half-life of TIP60. Further study found that HDAC3 colocalized with TIP60 both in the nucleus and the cytoplasm, which could be the reason why HDAC3 can stabilize TIP60. The deacetylation of TIP60 by both SIRT1 and HDAC3 reduces apoptosis induced by DNA damage. Knockdown of HDAC3 in cells increased TIP60 acetylation levels and increased apoptosis after DNA damage. Together, our findings provide a better understanding of TIP60 regulation mechanisms, which is a significant basis for further studies of its cellular functions. PMID:25301942

  4. Molecular epidemiology of camel trypanosomiasis based on ITS1 rDNA and RoTat 1.2 VSG gene in the Sudan

    PubMed Central

    2011-01-01

    Background Internal transcribed spacer one (ITS1) of the ribosomal DNA is known to be a suitable target for PCR-based detection of trypanosomes. The analysis of this region provides a multi-species-specific diagnosis by a single PCR. Using ITS1 primer-based PCR, a cross sectional study was carried out in the period from September to November 2009 on samples collected from 687 camels from geographically distinct zones in the Sudan to detect all possible African trypanosomes, which can infect camels. Results The results showed that all PCR-positive camels were infected with a single parasite species; Trypanosoma evansi. The highest prevalence, 57.1% (117/205), was observed in the Butana plains of mid-Eastern Sudan and the lowest, 6.0% (4/67), was in the Umshadeeda eastern part of White Nile State. In another experiment, the RoTat 1.2 gene encoding the variable surface glycoprotein (VSG) of T. evansi was analyzed for its presence or absence by a polymerase chain reaction (PCR) using T. evansi species-specific primers. The study showed that the RoTat 1.2 VSG gene was absent in thirteen out of thirty T. evansi-positive samples. Conclusions It is concluded that camel trypanosomiasis in Sudan is apparently caused by a single parasite species T. evansi and there were no other typanosomes species detected. In addition, the disease is highly prevalent in the country, which strengthens the need to change control policies and institute measures that help prevent the spread of the parasite. To our knowledge, this is the first molecular diagnosis report, which gives a picture of camel trypanosomiasis covering large geographical areas in Sudan. PMID:21375725

  5. Early Postnatal Manganese Exposure Causes Lasting Impairment of Selective and Focused Attention and Arousal Regulation in Adult Rats.

    PubMed

    Beaudin, Stephane A; Strupp, Barbara J; Strawderman, Myla; Smith, Donald R

    2017-02-01

    Studies in children and adolescents have associated early developmental manganese (Mn) exposure with inattention, impulsivity, hyperactivity, and oppositional behaviors, but causal inferences are precluded by the correlational nature of the data and generally limited control for potential confounders. To determine whether early postnatal oral Mn exposure causes lasting attentional and impulse control deficits in adulthood, and whether continued lifelong Mn exposure exacerbates these effects, using a rat model of environmental Mn exposure. Neonates were exposed orally to 0, 25 or 50 mg Mn/kg/day during early postnatal life (PND 1-21) or throughout life from PND 1 until the end of the study. In adulthood, the animals were tested on a series of learning and attention tasks using the five-choice serial reaction time task. Early postnatal Mn exposure caused lasting attentional dysfunction due to impairments in attentional preparedness, selective attention, and arousal regulation, whereas associative ability (learning) and impulse control were spared. The presence and severity of these deficits varied with the dose and duration of Mn exposure. This study is the first to show that developmental Mn exposure can cause lasting impairments in focused and selective attention and arousal regulation, and to identify the specific nature of the impairments. Given the importance of attention and arousal regulation in cognitive functioning, these findings substantiate concerns about the adverse effects of developmental Mn exposure in humans. Citation: Beaudin SA, Strupp BJ, Strawderman M, Smith DR. 2017. Early postnatal manganese exposure causes lasting impairment of selective and focused attention and arousal regulation in adult rats. Environ Health Perspect 125:230-237; http://dx.doi.org/10.1289/EHP258.

  6. Early Postnatal Manganese Exposure Causes Lasting Impairment of Selective and Focused Attention and Arousal Regulation in Adult Rats

    PubMed Central

    Beaudin, Stephane A.; Strupp, Barbara J.; Strawderman, Myla; Smith, Donald R.

    2016-01-01

    Background: Studies in children and adolescents have associated early developmental manganese (Mn) exposure with inattention, impulsivity, hyperactivity, and oppositional behaviors, but causal inferences are precluded by the correlational nature of the data and generally limited control for potential confounders. Objectives: To determine whether early postnatal oral Mn exposure causes lasting attentional and impulse control deficits in adulthood, and whether continued lifelong Mn exposure exacerbates these effects, using a rat model of environmental Mn exposure. Methods: Neonates were exposed orally to 0, 25 or 50 mg Mn/kg/day during early postnatal life (PND 1–21) or throughout life from PND 1 until the end of the study. In adulthood, the animals were tested on a series of learning and attention tasks using the five-choice serial reaction time task. Results: Early postnatal Mn exposure caused lasting attentional dysfunction due to impairments in attentional preparedness, selective attention, and arousal regulation, whereas associative ability (learning) and impulse control were spared. The presence and severity of these deficits varied with the dose and duration of Mn exposure. Conclusions: This study is the first to show that developmental Mn exposure can cause lasting impairments in focused and selective attention and arousal regulation, and to identify the specific nature of the impairments. Given the importance of attention and arousal regulation in cognitive functioning, these findings substantiate concerns about the adverse effects of developmental Mn exposure in humans. Citation: Beaudin SA, Strupp BJ, Strawderman M, Smith DR. 2017. Early postnatal manganese exposure causes lasting impairment of selective and focused attention and arousal regulation in adult rats. Environ Health Perspect 125:230–237; http://dx.doi.org/10.1289/EHP258 PMID:27384154

  7. HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System

    PubMed Central

    Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

    2015-01-01

    In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection. PMID:25613135

  8. Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

    PubMed Central

    Tan, Nguan-Soon; Shaw, Natacha S.; Vinckenbosch, Nicolas; Liu, Peng; Yasmin, Rubina; Desvergne, Béatrice; Wahli, Walter; Noy, Noa

    2002-01-01

    Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID:12077340

  9. Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion.

    PubMed

    Xu, Jie; Qin, Xinghua; Cai, Xiaoqing; Yang, Lu; Xing, Yuan; Li, Jun; Zhang, Lihua; Tang, Ying; Liu, Jiankang; Zhang, Xing; Gao, Feng

    2015-02-01

    c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Using generalized linear models to estimate selectivity from short-term recoveries of tagged red drum Sciaenops ocellatus: Effects of gear, fate, and regulation period

    USGS Publications Warehouse

    Bacheler, N.M.; Hightower, J.E.; Burdick, S.M.; Paramore, L.M.; Buckel, J.A.; Pollock, K.H.

    2010-01-01

    Estimating the selectivity patterns of various fishing gears is a critical component of fisheries stock assessment due to the difficulty in obtaining representative samples from most gears. We used short-term recoveries (n = 3587) of tagged red drum Sciaenops ocellatus to directly estimate age- and length-based selectivity patterns using generalized linear models. The most parsimonious models were selected using AIC, and standard deviations were estimated using simulations. Selectivity of red drum was dependent upon the regulation period in which the fish was caught, the gear used to catch the fish (i.e., hook-and-line, gill nets, pound nets), and the fate of the fish upon recovery (i.e., harvested or released); models including all first-order interactions between main effects outperformed models without interactions. Selectivity of harvested fish was generally dome-shaped and shifted toward larger, older fish in response to regulation changes. Selectivity of caught-and-released red drum was highest on the youngest and smallest fish in the early and middle regulation periods, but increased on larger, legal-sized fish in the late regulation period. These results suggest that catch-and-release mortality has consistently been high for small, young red drum, but has recently become more common in larger, older fish. This method of estimating selectivity from short-term tag recoveries is valuable because it is simpler than full tag-return models, and may be more robust because yearly fishing and natural mortality rates do not need to be modeled and estimated. ?? 2009 Elsevier B.V.

  11. Influence of maternal depression on children's brooding rumination: Moderation by CRHR1 TAT haplotype.

    PubMed

    Woody, Mary L; Kudinova, Anastacia Y; McGeary, John E; Knopik, Valerie S; Palmer, Rohan H C; Gibb, Brandon E

    2016-01-01

    There is growing evidence that brooding rumination plays a key role in the intergenerational transmission of major depressive disorder (MDD) and may be an endophenotype for depression risk. However, less is known about the mechanisms underlying this role. Therefore, the goal of the current study was to examine levels of brooding in children of mothers with a history of MDD (n = 129) compared to children of never depressed mothers (n = 126) and to determine whether the variation in a gene known to influence hypothalamic-pituitary-adrenal axis functioning--corticotropin-releasing hormone receptor 1 (CRHR1)--would moderate the link between maternal MDD and children's levels of brooding. We predicted children of mothers with a history of MDD would exhibit higher levels of brooding than children of mothers with no lifetime depression history but that this link would be stronger among children carrying no copies of the protective CRHR1 TAT haplotype. Our results supported these hypotheses and suggest that the development of brooding among children of depressed mothers, particularly children without the protective CRHR1 haplotype, may serve as an important mechanism of risk for the intergenerational transmission of depression.

  12. p53 Specifically Binds Triplex DNA In Vitro and in Cells

    PubMed Central

    Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

    2016-01-01

    Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed. PMID:27907175

  13. 49 CFR 17.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false What procedures apply to the selection of programs and activities under these regulations? 17.6 Section 17.6 Transportation Office of the Secretary of Transportation INTERGOVERNMENTAL REVIEW OF DEPARTMENT OF TRANSPORTATION PROGRAMS AND ACTIVITIES § 17.6 What...

  14. 28 CFR 30.6 - What procedures apply to the selection of programs and activities under these regulations?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false What procedures apply to the selection of programs and activities under these regulations? 30.6 Section 30.6 Judicial Administration DEPARTMENT OF... consult with local elected officials. (b) Each state that adopts a process shall notify the Attorney...

  15. Identification of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection

    PubMed Central

    McDonough, EmilyKate; Lazinski, David W.; Camilli, Andrew

    2014-01-01

    Summary Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment. PMID:24673931

  16. Identification of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection.

    PubMed

    McDonough, Emilykate; Lazinski, David W; Camilli, Andrew

    2014-04-01

    Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment. © 2014 John Wiley & Sons Ltd.

  17. Customising turnaround time indicators to requesting clinician: a 10-year study through balanced scorecard indicators.

    PubMed

    Salinas, Maria; López-Garrigós, Maite; Santo-Quiles, Ana; Gutierrez, Mercedes; Lugo, Javier; Lillo, Rosa; Leiva-Salinas, Carlos

    2014-09-01

    The purpose of this study is, first to present a 10-year monitoring of postanalytical turnaround time (TAT) adapted to different clinicians and patient situations, second to evaluate and analyse the indicators results during that period of time, and finally to show a synthetic appropriate indicator to be included in the balanced scorecard management system. TAT indicator for routine samples was devised as the percentage of certain key tests that were verified before a specific time on the phlebotomy day. A weighted mean synthetic indicator was also designed. They were calculated for inpatients at 15:00 and 12:00 and for primary care patients only at 15:00. The troponin TAT of emergency department patients, calculated as the difference between the troponin verification and registration time, was selected as the stat laboratory TAT indicator. The routine and stat TAT improved along the 10-year study period. The synthetic indicator showed the same trend. The implementation of systematic and continuous monitoring over years, promoted a continuous improvement in TAT which will probably benefit patient outcome and safety.

  18. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-08-16

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

  19. Regulating self-selection into private health insurance in Chile and the United States.

    PubMed

    Vargas Bustamante, Arturo; Méndez, Claudio A

    2016-07-01

    In the 1980s, Chile adopted a mixed (public and private) model for health insurance coverage similar to the one recently outlined by the Affordable Care Act in the United States (US). In such a system, a mix of public and private health plans offer nearly universal coverage using a combined approach of managed competition and subsidies for low-income individuals. This paper uses a "most different" case study design to compare policies implemented in Chile and the US to address self-selection into private insurance. We argue that the implementation of a mixed health insurance system in Chile without the appropriate regulations was complex, and it generated a series of inequities and perverse incentives. The comparison of Chile and the US healthcare reforms examines the different approaches that both countries have used to manage economic competition, address health insurance self-selection and promote solidarity. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  20. α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.

    PubMed

    Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

    2016-08-15

    Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. © 2016 Li, Miao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. Tuning Riboswitch Regulation through Conformational Selection

    PubMed Central

    Wilson, Ross C.; Smith, Angela M.; Fuchs, Ryan T.; Kleckner, Ian R.; Henkin, Tina M.; Foster, Mark P.

    2010-01-01

    SUMMARY The SMK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the SMK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound and ligand-free states. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element. PMID:21075119

  2. Distinct phosphodiesterase 5A-containing compartments allow selective regulation of cGMP-dependent signalling in human arterial smooth muscle cells.

    PubMed

    Wilson, Lindsay S; Guo, Manhong; Umana, M Bibiana; Maurice, Donald H

    2017-08-01

    Cyclic GMP (cGMP) translates and integrates much of the information encoded by nitric oxide (NO · ) and several natriuretic peptides, including the atrial natriuretic peptide (ANP). Previously, we reported that integration of a cGMP-specific cyclic nucleotide phosphodiesterase, namely phosphodiesterase 5A (PDE5A), into a protein kinase G (PKG)- and inositol-1,4,5-trisphosphate receptor (IP 3 R)-containing endoplasmic reticulum (ER) signalosome allows localized control of PDE5A activity and of PKG-dependent inhibition of IP 3 -mediated release of ER Ca 2+ in human platelets. Herein, we report that PDE5A integrates into an analogous signalosome in human arterial smooth muscle cells (HASMC), wherein it regulates muscarinic agonist-dependent Ca 2+ release and is activated selectively by PKG-dependent phosphorylation. In addition, we report that PDE5A also regulates HASMC functions via events independent of PKG, but rather through actions coordinated by competitive cGMP-mediated inhibition of cAMP hydrolysis by the so-called cGMP-inhibited cAMP PDE, namely phosphodiesterase 3A (PDE3A). Indeed, we show that ANP increases both cGMP and cAMP levels in HASMC and promotes phosphorylation of vasodilator-stimulated phospho-protein (VASP) at each the PKG and PKA phospho-acceptor sites. Since selective inhibition of PDE5 decreased DNA synthesis and chemotaxis of HASMC, and that PDE3A knockdown obviated these effects, our findings are consistent with a role for a PDE5A-PDE3A-PKA axis in their regulation. Our findings provide insight into the existence of distinct "pools" of PDE5A in HASMC and support the idea that these discrete compartments regulate distinct cGMP-dependent events. As a corollary, we suggest that it may be possible to target these distinct PDE5A-regulated pools and in so-doing differentially impact selected cGMP-regulated functions in these cells. Copyright © 2017. Published by Elsevier Inc.

  3. Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

    PubMed Central

    Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

    2015-01-01

    Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

  4. 7 CFR 946.25 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 946.25 Section 946.25 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Order Regulating Handling Administrative Committee § 946.25 Selection. (a) Persons selected as committee...

  5. 7 CFR 993.26 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 993.26 Section 993.26 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Order Regulating Handling Prune Marketing Committee § 993.26 Selection. Selection of members of the...

  6. 7 CFR 966.26 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 966.26 Section 966.26 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Regulating Handling Committee § 966.26 Selection. The Secretary shall select initially 3 members of the...

  7. 7 CFR 932.27 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 932.27 Section 932.27 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Regulating Handling Olive Administrative Committee § 932.27 Selection. Selection of members of the committee...

  8. Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

    PubMed

    Ueno, Akiko; Omori, Yoshihiro; Sugita, Yuko; Watanabe, Satoshi; Chaya, Taro; Kozuka, Takashi; Kon, Tetsuo; Yoshida, Satoyo; Matsushita, Kenji; Kuwahara, Ryusuke; Kajimura, Naoko; Okada, Yasushi; Furukawa, Takahisa

    2018-03-27

    In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Laboratory-based clinical audit as a tool for continual improvement: an example from CSF chemistry turnaround time audit in a South-African teaching hospital.

    PubMed

    Imoh, Lucius C; Mutale, Mubanga; Parker, Christopher T; Erasmus, Rajiv T; Zemlin, Annalise E

    2016-01-01

    Timeliness of laboratory results is crucial to patient care and outcome. Monitoring turnaround times (TAT), especially for emergency tests, is important to measure the effectiveness and efficiency of laboratory services. Laboratory-based clinical audits reveal opportunities for improving quality. Our aim was to identify the most critical steps causing a high TAT for cerebrospinal fluid (CSF) chemistry analysis in our laboratory. A 6-month retrospective audit was performed. The duration of each operational phase across the laboratory work flow was examined. A process-mapping audit trail of 60 randomly selected requests with a high TAT was conducted and reasons for high TAT were tested for significance. A total of 1505 CSF chemistry requests were analysed. Transport of samples to the laboratory was primarily responsible for the high average TAT (median TAT = 170 minutes). Labelling accounted for most delays within the laboratory (median TAT = 71 minutes) with most delays occurring after regular work hours (P < 0.05). CSF chemistry requests without the appropriate number of CSF sample tubes were significantly associated with delays in movement of samples from the labelling area to the technologist's work station (caused by a preference for microbiological testing prior to CSF chemistry). A laboratory-based clinical audit identified sample transportation, work shift periods and use of inappropriate CSF sample tubes as drivers of high TAT for CSF chemistry in our laboratory. The results of this audit will be used to change pre-analytical practices in our laboratory with the aim of improving TAT and customer satisfaction.

  10. Laboratory-based clinical audit as a tool for continual improvement: an example from CSF chemistry turnaround time audit in a South-African teaching hospital

    PubMed Central

    Imoh, Lucius C; Mutale, Mubanga; Parker, Christopher T; Erasmus, Rajiv T; Zemlin, Annalise E

    2016-01-01

    Introduction Timeliness of laboratory results is crucial to patient care and outcome. Monitoring turnaround times (TAT), especially for emergency tests, is important to measure the effectiveness and efficiency of laboratory services. Laboratory-based clinical audits reveal opportunities for improving quality. Our aim was to identify the most critical steps causing a high TAT for cerebrospinal fluid (CSF) chemistry analysis in our laboratory. Materials and methods A 6-month retrospective audit was performed. The duration of each operational phase across the laboratory work flow was examined. A process-mapping audit trail of 60 randomly selected requests with a high TAT was conducted and reasons for high TAT were tested for significance. Results A total of 1505 CSF chemistry requests were analysed. Transport of samples to the laboratory was primarily responsible for the high average TAT (median TAT = 170 minutes). Labelling accounted for most delays within the laboratory (median TAT = 71 minutes) with most delays occurring after regular work hours (P < 0.05). CSF chemistry requests without the appropriate number of CSF sample tubes were significantly associated with delays in movement of samples from the labelling area to the technologist’s work station (caused by a preference for microbiological testing prior to CSF chemistry). Conclusion A laboratory-based clinical audit identified sample transportation, work shift periods and use of inappropriate CSF sample tubes as drivers of high TAT for CSF chemistry in our laboratory. The results of this audit will be used to change pre-analytical practices in our laboratory with the aim of improving TAT and customer satisfaction. PMID:27346964

  11. Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

    PubMed

    Santoso, D; Thornburg, R

    2000-08-01

    We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

  12. Role of the p55-gamma subunit of PI3K in ALK-induced cell migration: RNAi-based selection of cell migration regulators.

    PubMed

    Seo, Minchul; Kim, Jong-Heon; Suk, Kyoungho

    2017-05-04

    Recently, unbiased functional genetic selection identified novel cell migration-regulating genes. This RNAi-based functional selection was performed using 63,996 pooled lentiviral shRNAs targeting 21,332 mouse genes. After five rounds of selection using cells with accelerated or impaired migration, shRNAs were retrieved and identified by half-hairpin barcode sequencing using cells with the selected phenotypes. This selection process led to the identification of 29 novel cell migration regulators. One of these candidates, anaplastic lymphoma kinase (ALK), was further investigated. Subsequent studies revealed that ALK promoted cell migration through the PI3K-AKT pathway via the p55γ regulatory subunit of PI3K, rather than more commonly used p85 subunit. Western blot and immunohistochemistry studies using mouse brain tissues revealed similar temporal expression patterns of ALK, phospho-p55γ, and phospho-AKT during different stages of development. These data support an important role for the p55γ subunit of PI3K in ALK-induced cell migration during brain development.

  13. Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

    PubMed

    T, Vinutha; Bansal, Navita; Kumari, Khushboo; Prashat G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, S K; Sachdev, Archana; Praveen, Shelly

    2017-12-20

    Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

  14. Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

    PubMed

    Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

    2013-01-14

    The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity.

    PubMed

    Chien, Li-Ting; Hartzell, H Criss

    2008-11-01

    Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

  16. High throughput selection of novel plant growth regulators: Assessing the translatability of small bioactive molecules from Arabidopsis to crops.

    PubMed

    Rodriguez-Furlán, Cecilia; Miranda, Giovanna; Reggiardo, Martín; Hicks, Glenn R; Norambuena, Lorena

    2016-04-01

    Plant growth regulators (PGRs) have become an integral part of agricultural and horticultural practices. Accordingly, there is an increased demand for new and cost-effective products. Nevertheless, the market is limited by insufficient innovation. In this context chemical genomics has gained increasing attention as a powerful approach addressing specific traits. Here is described the successful implementation of a highly specific, sensitive and efficient high throughput screening approach using Arabidopsis as a model. Using a combination of techniques, 10,000 diverse compounds were screened and evaluated for several important plant growth traits including root and leaf growth. The phenotype-based selection allowed the compilation of a collection of putative Arabidopsis growth regulators with a broad range of activities and specificities. A subset was selected for evaluating their bioactivity in agronomically valuable plants. Their validation as growth regulators in commercial species such as tomato, lettuce, carrot, maize and turfgrasses reinforced the success of the screening in Arabidopsis and indicated that small molecules activity can be efficiently translated to commercial species. Therefore, the chemical genomics approach in Arabidopsis is a promising field that can be incorporated in PGR discovery programs and has a great potential to develop new products that can be efficiently used in crops. Copyright © 2016. Published by Elsevier Ireland Ltd.

  17. Correlation between Interleukin-6 and Thrombin-Antithrombin III Complex Levels in Retinal Diseases.

    PubMed

    Ehrlich, Rita; Zahavi, Alon; Axer-Siegel, Ruth; Budnik, Ivan; Dreznik, Ayelet; Dahbash, Mor; Nisgav, Yael; Megiddo, Elinor; Kenet, Gili; Weinberger, Dov; Livnat, Tami

    2017-09-01

    This study aims to evaluate and correlate the levels of interleukin-6 (IL-6) and thrombin-antithrombin III complex (TAT) in the vitreous of patients with different vitreoretinal pathologies. Vitreous samples were collected from 78 patients scheduled for pars plana vitrectomy at a tertiary medical center. Patients were divided by the underlying vitreoretinal pathophysiology, as follows: macular hole (MH)/epiretinal membrane (ERM) (n = 26); rhegmatogenous retinal detachment (RRD) (n = 32); and proliferative diabetic retinopathy (PDR) (n = 20). Levels of IL-6 and TAT were measured by enzyme-linked immunosorbent assay and compared among the groups. A significant difference was found in the vitreal IL-6 and TAT levels between the MH/ERM group and both the PDR and RRD groups (P < 0.001 for all). Diabetes was associated with higher IL-6 levels in the RRD group. Different relationships between the IL-6 and TAT levels were revealed in patients with different ocular pathologies. Our results imply that variations in vitreal TAT level may be attributable not only to an inflammatory reaction or blood-retinal barrier breakdown, but also to intraocular tissue-dependent regulation of thrombin.

  18. A Study of the Relationship between Cognitive Emotion Regulation, Optimism, and Perceived Stress among Selected Teachers in Lutheran Schools

    ERIC Educational Resources Information Center

    Gliebe, Sudi Kate

    2012-01-01

    Problem: The problem of this study was to determine the relationship between perceived stress, as measured by the Perceived Stress Scale (PSS), and a specific set of predictor variables among selected teachers in Lutheran schools in the United States. These variables were cognitive emotion regulation strategies (positive reappraisal and…

  19. 7 CFR 923.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 923.23 Section 923.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... COUNTIES IN WASHINGTON Order Regulating Handling Administrative Body § 923.23 Selection. From the...

  20. 7 CFR 922.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 922.23 Section 922.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... IN WASHINGTON Order Regulating Handling Administrative Body § 922.23 Selection. From the nominations...

  1. HOW MUCH FAVORABLE SELECTION IS LEFT IN MEDICARE ADVANTAGE?

    PubMed Central

    PRICE, MARY; MCWILLIAMS, J. MICHAEL; HSU, JOHN; MCGUIRE, THOMAS G.

    2015-01-01

    The health economics literature contains two models of selection, one with endogenous plan characteristics to attract good risks and one with fixed plan characteristics; neither model contains a regulator. Medicare Advantage, a principal example of selection in the literature, is, however, subject to anti-selection regulations. Because selection causes economic inefficiency and because the historically favorable selection into Medicare Advantage plans increased government cost, the effectiveness of the anti-selection regulations is an important policy question, especially since the Medicare Advantage program has grown to comprise 30 percent of Medicare beneficiaries. Moreover, similar anti-selection regulations are being used in health insurance exchanges for those under 65. Contrary to earlier work, we show that the strengthened anti-selection regulations that Medicare introduced starting in 2004 markedly reduced government overpayment attributable to favorable selection in Medicare Advantage. At least some of the remaining selection is plausibly related to fixed plan characteristics of Traditional Medicare versus Medicare Advantage rather than changed selection strategies by Medicare Advantage plans. PMID:26389127

  2. Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

    PubMed Central

    Delos Santos, Ralph Christian; Bautista, Stephen; Lucarelli, Stefanie; Bone, Leslie N.; Dayam, Roya M.; Abousawan, John; Botelho, Roberto J.; Antonescu, Costin N.

    2017-01-01

    Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca2+, or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca2+- and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca2+ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes. PMID:28814502

  3. Multiple environmental factors regulate the expression of the carbohydrate-selective OprB porin of Pseudomonas aeruginosa.

    PubMed

    Adewoye, L O; Worobec, E A

    1999-12-01

    In response to low extracellular glucose concentration, Pseudomonas aeruginosa induces the expression of the outer membrane carbohydrate-selective OprB porin. The promoter region of the oprB gene was cloned into a lacZ transcriptional fusion vector, and the construct was mobilized into P. aeruginosa OprB-deficient strain, WW100, to evaluate additional environmental factors that influence OprB porin gene expression. Growth temperature, pH of the growth medium, salicylate concentration, and carbohydrate source were found to differentially influence porin expression. This expression pattern was compared to those of whole-cell [14C]glucose uptake under conditions of high osmolarity, ionicity, variable pH, growth temperatures, and carbohydrate source. These studies revealed that the high-affinity glucose transport genes are down-regulated by salicylic acid, differentially regulated by pH and temperature, and are specifically responsive to exogenous glucose induction.

  4. Fluoroorotic Acid-Selected Nicotiana plumbaginifolia Cell Lines with a Stable Thymine Starvation Phenotype Have Lost the Thymine-Regulated Transcriptional Program1

    PubMed Central

    Santoso, Djoko; Thornburg, Robert

    2000-01-01

    We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. PMID:10938367

  5. 48 CFR 1301.603-2 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Selection. 1301.603-2 Section 1301.603-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE GENERAL DEPARTMENT OF... 1301.603-2 Selection. In addition to the criteria set forth in FAR 1.603-2, selection of contracting...

  6. 7 CFR 905.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 905.23 Section 905.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... TANGELOS GROWN IN FLORIDA Order Regulating Handling Administrative Bodies § 905.23 Selection. (a) From the...

  7. 7 CFR 906.22 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 906.22 Section 906.22 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Committee § 906.22 Selection. (a) From District...

  8. Evolutionary dynamics on graphs: Efficient method for weak selection

    NASA Astrophysics Data System (ADS)

    Fu, Feng; Wang, Long; Nowak, Martin A.; Hauert, Christoph

    2009-04-01

    Investigating the evolutionary dynamics of game theoretical interactions in populations where individuals are arranged on a graph can be challenging in terms of computation time. Here, we propose an efficient method to study any type of game on arbitrary graph structures for weak selection. In this limit, evolutionary game dynamics represents a first-order correction to neutral evolution. Spatial correlations can be empirically determined under neutral evolution and provide the basis for formulating the game dynamics as a discrete Markov process by incorporating a detailed description of the microscopic dynamics based on the neutral correlations. This framework is then applied to one of the most intriguing questions in evolutionary biology: the evolution of cooperation. We demonstrate that the degree heterogeneity of a graph impedes cooperation and that the success of tit for tat depends not only on the number of rounds but also on the degree of the graph. Moreover, considering the mutation-selection equilibrium shows that the symmetry of the stationary distribution of states under weak selection is skewed in favor of defectors for larger selection strengths. In particular, degree heterogeneity—a prominent feature of scale-free networks—generally results in a more pronounced increase in the critical benefit-to-cost ratio required for evolution to favor cooperation as compared to regular graphs. This conclusion is corroborated by an analysis of the effects of population structures on the fixation probabilities of strategies in general 2×2 games for different types of graphs. Computer simulations confirm the predictive power of our method and illustrate the improved accuracy as compared to previous studies.

  9. HMGB1 modulation in pancreatic islets using a cell-permeable A-box fragment.

    PubMed

    Hwang, Yong Hwa; Kim, Min Jun; Lee, Yong-Kyu; Lee, Minhyung; Lee, Dong Yun

    2017-01-28

    Although pancreatic islet implantation is an attractive strategy for curing diabetes mellitus, implanted cells are immunologically eliminated due to early islet graft loss. One of main issues in early islet graft loss is the secretion of high-mobility group-box-1 (HMGB1) protein from the damaged islet cells, which is known as a cytokine-like factor. Therefore, regulating the activity of HMGB1 protein offers an alternative strategy for improving outcomes of islet cell therapy. To this end, we first demonstrated that HMGB1 protein could be bound to its A-box fragment (HMGB1 A-box) with higher binding affinity, resembling anti-HMGB1 antibody. To be used as a pharmaceutical protein ex vivo, TAT-labeled HMGB1 A-box-His 6 (TAT-HMGB1A) was structurally modified for cellular membrane penetration. TAT-HMGB1A significantly reduced secretion of endogenous HMGB1 protein through interaction in the cytosol without any damage to the viability or functionality of the islets. When TAT-HMGB1A-treated islets were implanted into diabetic nude mice, they completely cured diabetes, as evidenced by stable blood glucose level. TAT-HMGB1A treatment could also reduce the marginal islet mass needed to cure diabetes. Furthermore, TAT-HMGB1A positively protected xenotransplanted islets from xenogeneic immune reactions. Collectively, cell-penetrable TAT-HMGB1A could be used to modulate HMGB1 activity to increase successful outcomes of ex vivo pancreatic islet cell therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Electrical detection of the biological interaction of a charged peptide via gallium arsenide junction-field-effect transistors

    PubMed Central

    Lee, Kangho; Nair, Pradeep R.; Alam, Muhammad A.; Janes, David B.; Wampler, Heeyeon P.; Zemlyanov, Dmitry Y.; Ivanisevic, Albena

    2008-01-01

    GaAs junction-field-effect transistors (JFETs) are utilized to achieve label-free detection of biological interaction between a probe transactivating transcriptional activator (TAT) peptide and the target trans-activation-responsive (TAR) RNA. The TAT peptide is a short sequence derived from the human immunodeficiency virus-type 1 TAT protein. The GaAs JFETs are modified with a mixed adlayer of 1-octadecanethiol (ODT) and TAT peptide, with the ODT passivating the GaAs surface from polar ions in physiological solutions and the TAT peptide providing selective binding sites for TAR RNA. The devices modified with the mixed adlayer exhibit a negative pinch-off voltage (VP) shift, which is attributed to the fixed positive charges from the arginine-rich regions in the TAT peptide. Immersing the modified devices into a TAR RNA solution results in a large positive VP shift (>1 V) and a steeper subthreshold slope (∼80 mV∕decade), whereas “dummy” RNA induced a small positive VP shift (∼0.3 V) without a significant change in subthreshold slopes (∼330 mV∕decade). The observed modulation of device characteristics is analyzed with analytical modeling and two-dimensional numerical device simulations to investigate the electronic interactions between the GaAs JFETs and biological molecules. PMID:19484151

  11. Anion-Regulated Selective Generation of Cobalt Sites in Carbon: Toward Superior Bifunctional Electrocatalysis

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

    Wan, Gang; Yang, Ce; Zhao, Wanpeng

    The introduction of active transition metal sites (TMSs) in carbon enables the synthesis of noble-metal-free electrocatalysts for clean energy conversion applications, however, there are often multiple existing forms of TMSs, which are of different natures and catalytic models. Regulating the evolution of distinctive TMSs is highly desirable but remains challenging to date. Anions, as essential elements involved in the synthesis, have been totally neglected previously in the construction of TMSs. Herein, the effects of anions on the creation of different types of TMSs is investigated for the first time. It is found that the active cobalt-nitrogen sites tend to bemore » selectively constructed on the surface of N-doped carbon by using chloride, while metallic cobalt nanoparticles encased in protective graphite layers are the dominant forms of cobalt species with nitrate ions. The obtained catalysts demonstrate cobalt-sites-dependent activity for ORR and HER in acidic media. And the remarkably enhanced catalytic activities approaching that of benchmark Pt/C in acidic medium has been obtained on the catalyst dominated with cobalt-nitrogen sites, confirmed by the advanced spectroscopic . Our finding demonstrates a general paradigm of anion-regulated evolution of distinctive TMSs, providing a new pathway for enhancing performances of various targeted reactions related with TMSs.« less

  12. Genes Regulated by Vitamin D in Bone Cells Are Positively Selected in East Asians

    PubMed Central

    Chen, Yuan; Xue, Yali; Luiselli, Donata; Tyler-Smith, Chris; Pagani, Luca; Ayub, Qasim

    2015-01-01

    Vitamin D and folate are activated and degraded by sunlight, respectively, and the physiological processes they control are likely to have been targets of selection as humans expanded from Africa into Eurasia. We investigated signals of positive selection in gene sets involved in the metabolism, regulation and action of these two vitamins in worldwide populations sequenced by Phase I of the 1000 Genomes Project. Comparing allele frequency-spectrum-based summary statistics between these gene sets and matched control genes, we observed a selection signal specific to East Asians for a gene set associated with vitamin D action in bones. The selection signal was mainly driven by three genes CXXC finger protein 1 (CXXC1), low density lipoprotein receptor-related protein 5 (LRP5) and runt-related transcription factor 2 (RUNX2). Examination of population differentiation and haplotypes allowed us to identify several candidate causal regulatory variants in each gene. Four of these candidate variants (one each in CXXC1 and RUNX2 and two in LRP5) had a >70% derived allele frequency in East Asians, but were present at lower (20–60%) frequency in Europeans as well, suggesting that the adaptation might have been part of a common response to climatic and dietary changes as humans expanded out of Africa, with implications for their role in vitamin D-dependent bone mineralization and osteoporosis insurgence. We also observed haplotype sharing between East Asians, Finns and an extinct archaic human (Denisovan) sample at the CXXC1 locus, which is best explained by incomplete lineage sorting. PMID:26719974

  13. 7 CFR 915.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 915.23 Section 915.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Regulating Handling Administrative Body § 915.23 Selection. From the nominations made pursuant to § 915.22...

  14. 7 CFR 916.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 916.23 Section 916.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Regulating Handling Administrative Body § 916.23 Selection. From the nominations made pursuant to § 916.22...

  15. 7 CFR 929.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 929.23 Section 929.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... LONG ISLAND IN THE STATE OF NEW YORK Order Regulating Handling Administrative Body § 929.23 Selection...

  16. 48 CFR 36.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Selection criteria. 36.602-1 Section 36.602-1 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION SPECIAL... Selection criteria. (a) Agencies shall evaluate each potential contractor in terms of its— (1) Professional...

  17. 48 CFR 236.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Selection criteria. 236.602-1 Section 236.602-1 Federal Acquisition Regulations System DEFENSE ACQUISITION REGULATIONS SYSTEM... Architect-Engineer Services 236.602-1 Selection criteria. (a) Establish the evaluation criteria before...

  18. Socioemotional selectivity theory, aging, and health: the increasingly delicate balance between regulating emotions and making tough choices.

    PubMed

    Löckenhoff, Corinna E; Carstensen, Laura L

    2004-12-01

    After providing an introductory overview of socioemotional selectivity theory, we review empirical evidence for its basic postulates and consider the implications of the predicted cognitive and behavioral changes for physical health. The main assertion of socioemotional selectivity theory is that when boundaries on time are perceived, present-oriented goals related to emotional meaning are prioritized over future-oriented goals aimed at acquiring information and expanding horizons. Such motivational changes, which are strongly correlated with chronological age, systematically influence social preferences, social network composition, emotion regulation, and cognitive processing. On the one hand, there is considerable reason to believe that such changes are good for well-being and social adjustment. On the other hand, the very same motivational changes may limit health-related information-seeking and influence attention, memory, and decision-making such that positive material is favored over negative information. Grounding our arguments in socioemotional selectivity theory, we consider possible ways to tailor contexts such that disadvantages are avoided.

  19. How can the regulator show evidence of (no) risk selection in health insurance markets? Conceptual framework and empirical evidence.

    PubMed

    van de Ven, Wynand P M M; van Vliet, René C J A; van Kleef, Richard C

    2017-03-01

    If consumers have a choice of health plan, risk selection is often a serious problem (e.g., as in Germany, Israel, the Netherlands, the United States of America, and Switzerland). Risk selection may threaten the quality of care for chronically ill people, and may reduce the affordability and efficiency of healthcare. Therefore, an important question is: how can the regulator show evidence of (no) risk selection? Although this seems easy, showing such evidence is not straightforward. The novelty of this paper is two-fold. First, we provide a conceptual framework for showing evidence of risk selection in competitive health insurance markets. It is not easy to disentangle risk selection and the insurers' efficiency. We suggest two methods to measure risk selection that are not biased by the insurers' efficiency. Because these measures underestimate the true risk selection, we also provide a list of signals of selection that can be measured and that, in particular in combination, can show evidence of risk selection. It is impossible to show the absence of risk selection. Second, we empirically measure risk selection among the switchers, taking into account the insurers' efficiency. Based on 2-year administrative data on healthcare expenses and risk characteristics of nearly all individuals with basic health insurance in the Netherlands (N > 16 million) we find significant risk selection for most health insurers. This is the first publication of hard empirical evidence of risk selection in the Dutch health insurance market.

  20. Energy metabolism, fuel selection and body weight regulation

    PubMed Central

    Galgani, J; Ravussin, E

    2010-01-01

    Energy homeostasis is critical for the survival of species. Therefore, multiple and complex mechanisms have evolved to regulate energy intake and expenditure to maintain body weight. For weight maintenance, not only does energy intake have to match energy expenditure, but also macronutrient intake must balance macronutrient oxidation. However, this equilibrium seems to be particularly difficult to achieve in individuals with low fat oxidation, low energy expenditure, low sympathetic activity or low levels of spontaneous physical activity, as in addition to excess energy intake, all of these factors explain the tendency of some people to gain weight. Additionally, large variability in weight change is observed when energy surplus is imposed experimentally or spontaneously. Clearly, the data suggest a strong genetic influence on body weight regulation implying a normal physiology in an ‘obesogenic’ environment. In this study, we also review evidence that carbohydrate balance may represent the potential signal that regulates energy homeostasis by impacting energy intake and body weight. Because of the small storage capacity for carbohydrate and its importance for metabolism in many tissues and organs, carbohydrate balance must be maintained at a given level. This drive for balance may in turn cause increased energy intake when consuming a diet high in fat and low in carbohydrate. If sustained over time, such an increase in energy intake cannot be detected by available methods, but may cause meaningful increases in body weight. The concept of metabolic flexibility and its impact on body weight regulation is also presented. PMID:19136979

  1. High-yield nontoxic gene transfer through conjugation of the CM₁₈-Tat₁₁ chimeric peptide with nanosecond electric pulses.

    PubMed

    Salomone, Fabrizio; Breton, Marie; Leray, Isabelle; Cardarelli, Francesco; Boccardi, Claudia; Bonhenry, Daniel; Tarek, Mounir; Mir, Lluis M; Beltram, Fabio

    2014-07-07

    We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols.

  2. 48 CFR 1436.602-4 - Selection authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Selection authority. 1436.602-4 Section 1436.602-4 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR SPECIAL...-4 Selection authority. The HCA is authorized to serve as the designated selection authority. ...

  3. 48 CFR 1336.602-4 - Selection authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Selection authority. 1336.602-4 Section 1336.602-4 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE SPECIAL...-4 Selection authority. Each contracting office shall designate the selection authority based on the...

  4. Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1

    PubMed Central

    Schwertassek, Ulla; Balmer, Yves; Gutscher, Marcus; Weingarten, Lars; Preuss, Marc; Engelhard, Johanna; Winkler, Monique; Dick, Tobias P

    2007-01-01

    The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor–ligand signaling interactions can be selectively regulated by an extracellular redox catalyst. PMID:17557078

  5. 48 CFR 2936.602-4 - Selection Authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Selection Authority. 2936.602-4 Section 2936.602-4 Federal Acquisition Regulations System DEPARTMENT OF LABOR GENERAL... Selection Authority. The HCA is authorized to serve as the designated Selection Authority in accordance with...

  6. 48 CFR 2436.602-4 - Selection authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Selection authority. 2436.602-4 Section 2436.602-4 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN... Services 2436.602-4 Selection authority. (a) The final selection decision shall be made by the cognizant...

  7. NeuroD modulates opioid agonist-selective regulation of adult neurogenesis and contextual memory extinction.

    PubMed

    Zheng, Hui; Zhang, Yue; Li, Wen; Loh, Horace H; Law, Ping-Yee

    2013-04-01

    Addictive drugs, including opioids, modulate adult neurogenesis. In order to delineate the probable implications of neurogenesis on contextual memory associated with addiction, we investigated opioid agonist-selective regulation of neurogenic differentiation 1 (NeuroD) activities under the conditioned place preference (CPP) paradigm. Training mice with equivalent doses of morphine and fentanyl produced different CPP extinction rates without measurable differences in the CPP acquisition rate or magnitude. Fentanyl-induced CPP required much longer time for extinction than morphine-induced CPP. We observed a parallel decrease in NeuroD activities and neurogenesis after morphine-induced CPP, but not after fentanyl-induced CPP. Increasing NeuroD activities with NeuroD-lentivirus (nd-vir) injection at the dentate gyrus before CPP training reversed morphine-induced decreases in NeuroD activities and neurogenesis, and prolonged the time required for extinction of morphine-induced CPP. On the other hand, decreasing NeuroD activities via injection of miRNA-190-virus (190-vir) reversed the fentanyl effect on NeuroD and neurogenesis and shortened the time required for extinction of fentanyl-induced CPP. Another contextual memory task, the Morris Water Maze (MWM), was affected similarly by alteration of NeuroD activities. The reduction in NeuroD activities either by morphine treatment or 190-vir injection decreased MWM task retention, while the increase in NeuroD activities by nd-vir prolonged MWM task retention. Thus, by controlling NeuroD activities, opioid agonists differentially regulate adult neurogenesis and subsequent contextual memory retention. Such drug-related memory regulation could have implications in eventual context-associated relapse.

  8. Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

    PubMed

    Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

    2013-12-01

    Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

  9. Myopic (HD-PTP, PTPN23) selectively regulates synaptic neuropeptide release.

    PubMed

    Bulgari, Dinara; Jha, Anupma; Deitcher, David L; Levitan, Edwin S

    2018-02-13

    Neurotransmission is mediated by synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs) and small-molecule transmitter-containing small synaptic vesicles (SSVs). Exocytosis of both vesicle types depends on Ca 2+ and shared secretory proteins. Here, we show that increasing or decreasing expression of Myopic (mop, HD-PTP, PTPN23), a Bro1 domain-containing pseudophosphatase implicated in neuronal development and neuropeptide gene expression, increases synaptic neuropeptide stores at the Drosophila neuromuscular junction (NMJ). This occurs without altering DCV content or transport, but synaptic DCV number and age are increased. The effect on synaptic neuropeptide stores is accounted for by inhibition of activity-induced Ca 2+ -dependent neuropeptide release. cAMP-evoked Ca 2+ -independent synaptic neuropeptide release also requires optimal Myopic expression, showing that Myopic affects the DCV secretory machinery shared by cAMP and Ca 2+ pathways. Presynaptic Myopic is abundant at early endosomes, but interaction with the endosomal sorting complex required for transport III (ESCRT III) protein (CHMP4/Shrub) that mediates Myopic's effect on neuron pruning is not required for control of neuropeptide release. Remarkably, in contrast to the effect on DCVs, Myopic does not affect release from SSVs. Therefore, Myopic selectively regulates synaptic DCV exocytosis that mediates peptidergic transmission at the NMJ.

  10. The effect of potential electronic nicotine delivery system regulations on nicotine product selection.

    PubMed

    Pesko, Michael F; Kenkel, Donald S; Wang, Hua; Hughes, Jenna M

    2016-04-01

    To estimate the effect of potential regulations of electronic nicotine delivery systems (ENDS) among adult smokers, including increasing taxes, reducing flavor availability and adding warning labels communicating various levels of risk. We performed a discrete choice experiment (DCE) among a national sample of 1200 adult smokers. We examined heterogeneity in policy responses by age, cigarette quitting interest and current ENDS use. Our experiment overlapped January 2015 by design, providing exogenous variation in cigarette quitting interest from New Year resolutions. KnowledgePanel, an online panel of recruited respondents. A total of 1200 adult smokers from the United States. Hypothetical purchase choice of cigarettes, nicotine replacement therapy and a disposable ENDS. Increasing ENDS prices from $3 to $6 was associated with a 13.6 percentage point reduction in ENDS selection (P < 0.001). Restricting flavor availability in ENDS to tobacco and menthol was associated with a 2.1 percentage point reduction in ENDS selection (P < 0.001). The proposed Food and Drug Administration (FDA) warning label was associated with a 1.1 percentage point reduction in ENDS selection (P < 0.05) and the MarkTen warning label with a 5.1 percentage point reduction (P < 0.001). We estimated an ENDS price elasticity of -1.8 (P < 0.001) among adult smokers. Statistically significant interaction terms (P < 0.001) imply that price responsiveness was higher among adult smokers 18-24 years of age, smokers who have vaped over the last month and smokers with above the median quitting interest. Young adult smokers were 3.7 percentage points more likely to choose ENDS when multiple flavors were available than older adults (P < 0.001). Young adult smokers and those with above the median cigarette quitting interest were also more likely to reduce cigarette selection and increase ENDS selection in January 2015 (P < 0.001), potentially in response to New Year's resolutions to quit

  11. Glioma targeted delivery strategy of doxorubicin-loaded liposomes by dual-ligand modification.

    PubMed

    Han, Wei; Yin, Guangfu; Pu, Ximing; Chen, Xianchun; Liao, Xiaoming; Huang, Zhongbing

    2017-10-01

    The blood-brain barrier (BBB) is the protective parclose of brain safety, but it is also the main obstacle of the drug delivery to cerebral parenchyma, which hamper therapy for brain diseases. In this work, a glioma targeted drug delivery system was developed through loading doxorubicin into Angiopep-2 and TAT peptide dual-modified liposomes (DOX-TAT-Ang-LIP). Low-density lipoprotein receptor-related protein-1 (LRP1) was one receptor overexpressed on both BBB and glioma cytomembranes. Angiopep-2, a specific ligand of LRP1, exhibited high LRP1 binding efficiency. Additionally, TAT could penetrate through cell membranes without selectivity via an unsaturated pathway. To avoid the receptor saturation of Angiopep-2, TAT was also conjugated on the surface of liposomes, providing that the liposomes not only have effective BBB penetrating effect, but also have the glioma targeting function. The prepared DOX liposomes appeared good stability and narrow dispersity in serum with a diameter of 90 nm, and exhibited sustained DOX release behaviors. The conjunctions of Angiopep-2 and TAT were confirmed by 1 H NMR spectra. The BBB model, cellular uptake observations, antiproliferation study, and the cell ultrastructure analyses suggested that DOX-TAT-Ang-LIP could not only penetrate through BBB via transcytosis, but also concentrate in glioma, then enter into glioma cells and finally result in the necrosis of glioma cells.

  12. Inhibition of mitochondrial fragmentation diminishes Huntington’s disease–associated neurodegeneration

    PubMed Central

    Guo, Xing; Disatnik, Marie-Helene; Monbureau, Marie; Shamloo, Mehrdad; Mochly-Rosen, Daria; Qi, Xin

    2013-01-01

    Huntington’s disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell–derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell–derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT–like inhibitor may prevent or slow the progression of HD. PMID:24231356

  13. Regulation of Yeast H+-ATPase by Protein Kinases Belonging to a Family Dedicated to Activation of Plasma Membrane Transporters

    PubMed Central

    Goossens, Alain; de la Fuente, Natalia; Forment, Javier; Serrano, Ramon; Portillo, Francisco

    2000-01-01

    The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H+-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters). PMID:11003661

  14. Self-regulation and selective exposure: the impact of depleted self-regulation resources on confirmatory information processing.

    PubMed

    Fischer, Peter; Greitemeyer, Tobias; Frey, Dieter

    2008-03-01

    In the present research, the authors investigated the impact of self-regulation resources on confirmatory information processing, that is, the tendency of individuals to systematically prefer standpoint-consistent information to standpoint-inconsistent information in information evaluation and search. In 4 studies with political and economic decision-making scenarios, it was consistently found that individuals with depleted self-regulation resources exhibited a stronger tendency for confirmatory information processing than did individuals with nondepleted self-regulation resources. Alternative explanations based on processes of ego threat, cognitive load, and mood were ruled out. Mediational analyses suggested that individuals with depleted self-regulation resources experienced increased levels of commitment to their own standpoint, which resulted in increased confirmatory information processing. In sum, the impact of ego depletion on confirmatory information search seems to be more motivational than cognitive in nature.

  15. Isolation gowns in health care settings: Laboratory studies, regulations and standards, and potential barriers of gown selection and use

    PubMed Central

    Kilinc Balci, F. Selcen

    2016-01-01

    Although they play an important role in infection prevention and control, textile materials and personal protective equipment (PPE) used in health care settings are known to be one of the sources of cross-infection. Gowns are recommended to prevent transmission of infectious diseases in certain settings; however, laboratory and field studies have produced mixed results of their efficacy. PPE used in health care is regulated as either class I (low risk) or class II (intermediate risk) devices in the United States. Many organizations have published guidelines for the use of PPE, including isolation gowns, in health care settings. In addition, the Association for the Advancement of Medical Instrumentation published a guidance document on the selection of gowns and a classification standard on liquid barrier performance for both surgical and isolation gowns. However, there is currently no existing standard specific to isolation gowns that considers not only the barrier resistance but also a wide array of end user desired attributes. As a result, infection preventionists and purchasing agents face several difficulties in the selection process, and end users have limited or no information on the levels of protection provided by isolation gowns. Lack of knowledge about the performance of protective clothing used in health care became more apparent during the 2014 Ebola epidemic. This article reviews laboratory studies, regulations, guidelines and standards pertaining to isolation gowns, characterization problems, and other potential barriers of isolation gown selection and use. PMID:26391468

  16. A Genetic Selection For Neurospora crassa Mutants Altered in Their Light Regulation of Transcription

    PubMed Central

    Navarro-Sampedro, Laura; Yanofsky, Charles; Corrochano, Luis M.

    2008-01-01

    Transcription of the Neurospora crassa gene con-10 is induced during conidiation and following exposure of vegetative mycelia to light, but light activation is transient due to photoadaptation. We describe mutational analyses of photoadaptation using a N. crassa strain bearing a translational fusion of con-10, including its regulatory region, to a selectable bacterial gene conferring hygromycin resistance (hph). Growth of this strain was sensitive to hygromycin, upon continuous culture in the light. Five mutants were isolated that were resistant to hygromycin when cultured under constant light. Three mutant strains displayed elevated, sustained accumulation of con-10∷hph mRNA during continued light exposure, suggesting that they bear mutations that reduce or eliminate the presumed light-dependent repression mechanism that blocks con-10 transcription upon prolonged illumination. These mutations altered photoadaptation for only a specific group of genes (con-10 and con-6), suggesting that regulation of photoadaptation is relatively gene specific. The mutations increased light-dependent mRNA accumulation for genes al-1, al-2, and al-3, each required for carotenoid biosynthesis, resulting in a threefold increase in carotenoid accumulation following continuous light exposure. Identification of the altered gene or genes in these mutants may reveal novel proteins that participate in light regulation of gene transcription in fungi. PMID:18202366

  17. 48 CFR 1401.603-2 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Selection. 1401.603-2 Section 1401.603-2 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR GENERAL DEPARTMENT OF... Responsibilities 1401.603-2 Selection. COs, regardless of series or organizational placement, must be certified at...

  18. Enhancing students’ cognitive skill in Nguyen Tat Thanh high school Hanoi Vietnam through scientific learning material of static electricity

    NASA Astrophysics Data System (ADS)

    Priyanto, A.; Linuwih, S.; Aji, M. P.; Bich, D. D.

    2018-03-01

    Scientific learning material is still needed by students at Nguyen Tat Thanh High School (NTT), Hanoi Vietnam in order to enhance the students’ cognitive skill. Cognitive skill represents the level of students’ understanding to the particular material. Students’ cognitive skill can be improved by applying the learning material based on scientific approach as a treatment. The enhancement of students’ cognitive skill can be measured by analyzing the students’ test result collected before and after treatment. The analysis is focused to measure the enhancement or the sifted of cognitive aspects including remembering aspect (C1), understanding aspect (C2), applying aspect (C3), analyzing aspect (C4), and evaluating aspect (C5). According to the analysis the enhancement of cognitive aspects are 8.26% of remembering, 3.26% of understanding, 32.94% of applying, 21.74% of analyzing, and 21.74% of evaluating. The major enhancements are occured at applying, analyzing, and evaluating aspects. Therefore it can be concluded that students’ cognitive skill is enhanced by applying scientific learning material of static electricity.

  19. Tempo-spatially resolved cellular dynamics of human immunodeficiency virus transacting activator of transcription (Tat) peptide-modified nanocargos in living cells

    NASA Astrophysics Data System (ADS)

    Wei, Lin; Yang, Qiaoyu; Xiao, Lehui

    2014-08-01

    Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs

  20. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement.

    PubMed

    Altenhöfer, Sebastian; Radermacher, Kim A; Kleikers, Pamela W M; Wingler, Kirstin; Schmidt, Harald H H W

    2015-08-10

    Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition.

  1. Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium.

    PubMed

    Delos Santos, Ralph Christian; Bautista, Stephen; Lucarelli, Stefanie; Bone, Leslie N; Dayam, Roya M; Abousawan, John; Botelho, Roberto J; Antonescu, Costin N

    2017-10-15

    Clathrin-mediated endocytosis is a major regulator of cell-surface protein internalization. Clathrin and other proteins assemble into small invaginating structures at the plasma membrane termed clathrin-coated pits (CCPs) that mediate vesicle formation. In addition, epidermal growth factor receptor (EGFR) signaling is regulated by its accumulation within CCPs. Given the diversity of proteins regulated by clathrin-mediated endocytosis, how this process may distinctly regulate specific receptors is a key question. We examined the selective regulation of clathrin-dependent EGFR signaling and endocytosis. We find that perturbations of phospholipase Cγ1 (PLCγ1), Ca 2+ , or protein kinase C (PKC) impair clathrin-mediated endocytosis of EGFR, the formation of CCPs harboring EGFR, and EGFR signaling. Each of these manipulations was without effect on the clathrin-mediated endocytosis of transferrin receptor (TfR). EGFR and TfR were recruited to largely distinct clathrin structures. In addition to control of initiation and assembly of CCPs, EGF stimulation also elicited a Ca 2+ - and PKC-dependent reduction in synaptojanin1 recruitment to clathrin structures, indicating broad control of CCP assembly by Ca 2+ signals. Hence EGFR elicits PLCγ1-calcium signals to facilitate formation of a subset of CCPs, thus modulating its own signaling and endocytosis. This provides evidence for the versatility of CCPs to control diverse cellular processes. © 2017 Delos Santos et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand.

    PubMed

    Pichanun, Dalad; Munkongdee, Thongperm; Klamchuen, Sumonmaln; Butthep, Punnee; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

    2010-01-01

    Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.

  3. Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

    PubMed

    Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

    2017-02-13

    Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

  4. Closed-form solutions for linear regulator-design of mechanical systems including optimal weighting matrix selection

    NASA Technical Reports Server (NTRS)

    Hanks, Brantley R.; Skelton, Robert E.

    1991-01-01

    This paper addresses the restriction of Linear Quadratic Regulator (LQR) solutions to the algebraic Riccati Equation to design spaces which can be implemented as passive structural members and/or dampers. A general closed-form solution to the optimal free-decay control problem is presented which is tailored for structural-mechanical systems. The solution includes, as subsets, special cases such as the Rayleigh Dissipation Function and total energy. Weighting matrix selection is a constrained choice among several parameters to obtain desired physical relationships. The closed-form solution is also applicable to active control design for systems where perfect, collocated actuator-sensor pairs exist. Some examples of simple spring mass systems are shown to illustrate key points.

  5. Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana.

    PubMed

    Sandorf, Iris; Holländer-Czytko, Heike

    2002-11-01

    Coronatine-inducible tyrosine aminotransferase (TAT), which catalyses the transamination from tyrosine to p-hydroxyphenylpyruvate, is the first enzyme of a pathway leading via homogentisic acid to plastoquinone and tocopherols, the latter of which are known to be radical scavengers in plants. TAT can be also induced by the octadecanoids methyl jasmonate (MeJA) and methyl-12-oxophytodienoic acid (MeOPDA), as well as by wounding, high light, UV light and the herbicide oxyfluorfen. In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana (L.) Heynh., the jasmonate-deficient mutant delayed dehiscence (dde1) was used, in which the gene for 12-oxophytodienoic acid reductase 3 is disrupted. The amount of immunodetectable TAT was low. The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type. Treatment with MeOPDA, wounding and UV light, however, had hardly any effects. Tocopherol levels that showed considerable increases in the wild type after some treatments were much less affected in the mutant. However, starting levels of tocopherol were higher in non-induced dde1 than in the wild type. We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.

  6. 7 CFR 959.26 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 959.26 Section 959.26 Agriculture... Regulating Handling Committee § 959.26 Selection. The Secretary shall select members and respective alternates from districts established pursuant to § 959.24 or § 959.25. Initial selections shall be as...

  7. Self-Regulation among Youth in Four Western Cultures: Is There an Adolescence-Specific Structure of the Selection-Optimization-Compensation (SOC) Model?

    ERIC Educational Resources Information Center

    Gestsdottir, Steinunn; Geldhof, G. John; Paus, Tomáš; Freund, Alexandra M.; Adalbjarnardottir, Sigrun; Lerner, Jacqueline V.; Lerner, Richard M.

    2015-01-01

    We address how to conceptualize and measure intentional self-regulation (ISR) among adolescents from four cultures by assessing whether ISR (conceptualized by the SOC model of Selection, Optimization, and Compensation) is represented by three factors (as with adult samples) or as one "adolescence-specific" factor. A total of 4,057 14-…

  8. 48 CFR 2415.308 - Source selection decision.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Source selection decision. 2415.308 Section 2415.308 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN... document its selection recommendation(s) in a final written report. The final report shall include...

  9. Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes.

    PubMed

    Mancone, Carmine; Grimaldi, Alessio; Refolo, Giulia; Abbate, Isabella; Rozera, Gabriella; Benelli, Dario; Fimia, Gian Maria; Barnaba, Vincenzo; Tripodi, Marco; Piacentini, Mauro; Ciccosanti, Fabiola

    2017-01-01

    Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. RT-qPCR targeting the LTR region, gag , Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

  10. Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869▿

    PubMed Central

    Kikuchi, Yoshimi; Date, Masayo; Itaya, Hiroshi; Matsui, Kazuhiko; Wu, Long-Fei

    2006-01-01

    Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins. PMID:16997984

  11. 48 CFR 1436.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Selection criteria. 1436.602-1 Section 1436.602-1 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR SPECIAL...-1 Selection criteria. (a) The CO may include specific evaluation criteria to be used in the...

  12. 48 CFR 2936.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Selection criteria. 2936.602-1 Section 2936.602-1 Federal Acquisition Regulations System DEPARTMENT OF LABOR GENERAL... Selection criteria. HCAs are authorized to approve the use of design competition under the conditions in FAR...

  13. 48 CFR 436.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Selection criteria. 436.602-1 Section 436.602-1 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE SPECIAL... Selection criteria. The HCA is authorized to approve the use of design competition under the conditions in...

  14. 7 CFR 925.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 925.23 Section 925.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... SOUTHEASTERN CALIFORNIA Administrative Body § 925.23 Selection. The Secretary shall select members and...

  15. 7 CFR 958.22 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 958.22 Section 958.22 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Selection. The Secretary shall select committee members and alternates from the nominee lists submitted...

  16. Control of total GFP expression by alterations to the 3′ region nucleotide sequence

    PubMed Central

    2013-01-01

    Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found

  17. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor

    USDA-ARS?s Scientific Manuscript database

    We describe here the use of label-free wide selected-ion monitoring data-independent acquisition (WiSIM-DIA) to identify proteins that are involved in the formation of tomato (Solanum lycopersicum) fruit cuticles and that are regulated by the transcription factor CUTIN DEFICIENT2 (CD2). A spectral l...

  18. Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

    PubMed

    Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

    2017-01-01

    The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

  19. Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays

    PubMed Central

    Agosto-Arroyo, Emmanuel; Coshatt, Gina M.; Winokur, Thomas S.; Harada, Shuko; Park, Seung L.

    2017-01-01

    Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

  20. Genome-wide detection of selection signatures in Chinese indigenous Laiwu pigs revealed candidate genes regulating fat deposition in muscle.

    PubMed

    Chen, Minhui; Wang, Jiying; Wang, Yanping; Wu, Ying; Fu, Jinluan; Liu, Jian-Feng

    2018-05-18

    Currently, genome-wide scans for positive selection signatures in commercial breed have been investigated. However, few studies have focused on selection footprints of indigenous breeds. Laiwu pig is an invaluable Chinese indigenous pig breed with extremely high proportion of intramuscular fat (IMF), and an excellent model to detect footprint as the result of natural and artificial selection for fat deposition in muscle. In this study, based on GeneSeek Genomic profiler Porcine HD data, three complementary methods, F ST , iHS (integrated haplotype homozygosity score) and CLR (composite likelihood ratio), were implemented to detect selection signatures in the whole genome of Laiwu pigs. Totally, 175 candidate selected regions were obtained by at least two of the three methods, which covered 43.75 Mb genomic regions and corresponded to 1.79% of the genome sequence. Gene annotation of the selected regions revealed a list of functionally important genes for feed intake and fat deposition, reproduction, and immune response. Especially, in accordance to the phenotypic features of Laiwu pigs, among the candidate genes, we identified several genes, NPY1R, NPY5R, PIK3R1 and JAKMIP1, involved in the actions of two sets of neurons, which are central regulators in maintaining the balance between food intake and energy expenditure. Our results identified a number of regions showing signatures of selection, as well as a list of functionally candidate genes with potential effect on phenotypic traits, especially fat deposition in muscle. Our findings provide insights into the mechanisms of artificial selection of fat deposition and further facilitate follow-up functional studies.

  1. 48 CFR 636.602-1 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Selection criteria. 636.602-1 Section 636.602-1 Federal Acquisition Regulations System DEPARTMENT OF STATE SPECIAL CATEGORIES... Selection criteria. (b) The head of the contracting activity is the agency head's designee for the purpose...

  2. Selection methods regulate evolution of cooperation in digital evolution

    PubMed Central

    Lichocki, Paweł; Floreano, Dario; Keller, Laurent

    2014-01-01

    A key, yet often neglected, component of digital evolution and evolutionary models is the ‘selection method’ which assigns fitness (number of offspring) to individuals based on their performance scores (efficiency in performing tasks). Here, we study with formal analysis and numerical experiments the evolution of cooperation under the five most common selection methods (proportionate, rank, truncation-proportionate, truncation-uniform and tournament). We consider related individuals engaging in a Prisoner's Dilemma game where individuals can either cooperate or defect. A cooperator pays a cost, whereas its partner receives a benefit, which affect their performance scores. These performance scores are translated into fitness by one of the five selection methods. We show that cooperation is positively associated with the relatedness between individuals under all selection methods. By contrast, the change in the performance benefit of cooperation affects the populations’ average level of cooperation only under the proportionate methods. We also demonstrate that the truncation and tournament methods may introduce negative frequency-dependence and lead to the evolution of polymorphic populations. Using the example of the evolution of cooperation, we show that the choice of selection method, though it is often marginalized, can considerably affect the evolutionary dynamics. PMID:24152811

  3. Locating the Anion-selectivity Filter of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel

    PubMed Central

    Cheung, Min; Akabas, Myles H.

    1997-01-01

    The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineered cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by ∼25-fold and may involve the residues Arg347 and Arg352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter. PMID:9089437

  4. Initial assembly steps of a translocase for folded proteins

    PubMed Central

    Blümmel, Anne-Sophie; Haag, Laura A.; Eimer, Ekaterina; Müller, Matthias; Fröbel, Julia

    2015-01-01

    The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors. PMID:26068441

  5. 7 CFR 985.24 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 985.24 Section 985.24 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements....24 Selection. Committee members shall be selected by the Secretary from nominees submitted by the...

  6. 5 CFR 330.1105 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Selection. 330.1105 Section 330.1105 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS RECRUITMENT, SELECTION, AND... District of Columbia Department of Corrections § 330.1105 Selection. (a) If two or more individuals...

  7. Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria

    PubMed Central

    Sabrina Pankey, M; Foxall, Randi L; Ster, Ian M; Perry, Lauren A; Schuster, Brian M; Donner, Rachel A; Coyle, Matthew; Cooper, Vaughn S; Whistler, Cheryl A

    2017-01-01

    Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations. DOI: http://dx.doi.org/10.7554/eLife.24414.001 PMID:28447935

  8. Selecting "saviour siblings": reconsidering the regulation in Australia of pre-implantation genetic diagnosis in conjunction with tissue typing.

    PubMed

    Taylor-Sands, Michelle

    2007-05-01

    In recent years, pre-implantation genetic diagnosis (PGD) has been developed to enable the selection of a tissue type matched "saviour sibling" for a sick child. This article examines the current regulatory framework governing PGD in Australia. The availability of PGD in Australia to create a saviour sibling depends on the regulation of ART services by each State and Territory. The limitations on the use of PGD vary throughout Australia, according to the level of regulation of ART in each jurisdiction. This article considers the limitations on the use of PGD for tissue typing in Australia and argues that some of these should be removed for a more consistent national approach. In particular, the focus in ART legislation on the "paramount interests" of the child to be born is inappropriate for the application of tissue typing, which necessarily involves the interests of other family members.

  9. Selectivity as an Emotion Regulation Strategy: Lessons from Older Adults

    PubMed Central

    Sims, Tamara; Hogan, Candice; Carstensen, Laura

    2015-01-01

    Findings based on studies of daily life consistently associate older ages with relatively positive emotional experience, suggesting that older adults may regulate emotions more effectively than younger adults. Findings from laboratory studies are equivocal, however, with mixed evidence for age-related improvements in use of emotion regulatory strategies. In the current paper, we propose that findings may reflect a failure of laboratory-based experiments to capture the regulatory strategies that older people use in their everyday lives. We argue that the advantages older people have are likely due to antecedent emotion regulation as opposed to response-focused strategies. Understanding the regulatory approaches that older people actually use may inform developmental models of emotion regulation throughout adulthood as well as interventions for improving emotional experience across the life span. PMID:25914897

  10. 48 CFR 715.370 - Alternative source selection procedures.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Alternative source selection procedures. 715.370 Section 715.370 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Source Selection 715...

  11. Regulated Proteolysis in Bacteria.

    PubMed

    Mahmoud, Samar A; Chien, Peter

    2018-06-20

    Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.

  12. 7 CFR 945.24 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 945.24 Section 945.24 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... § 945.24 Selection. Members and alternates of the committee shall be selected by the Secretary on the...

  13. Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

    PubMed

    Kim, Jun Sub; Kim, Jae Gyu; Jeon, Chan Young; Won, Ha Young; Moon, Mi Young; Seo, Ji Yeon; Kim, Jong Il; Kim, Jaebong; Lee, Jae Yong; Choi, Soo Young; Park, Jinseu; Yoon Park, Jung Han; Ha, Kwon Soo; Kim, Pyeung Hyeun; Park, Jae Bong

    2005-12-31

    Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

  14. 48 CFR 1.603-2 - Selection.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ACQUISITION REGULATIONS SYSTEM Career Development, Contracting Authority, and Responsibilities 1.603-2 Selection. In selecting contracting officers, the appointing official shall consider the complexity and..., business acumen, judgment, character, and reputation. Examples of selection criteria include— (a...

  15. 5 CFR 330.1204 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Selection. 330.1204 Section 330.1204 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS RECRUITMENT, SELECTION, AND... Employees § 330.1204 Selection. (a) If two or more individuals apply for a vacancy and the hiring agency...

  16. 7 CFR 989.30 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 989.30 Section 989.30 Agriculture... CALIFORNIA Order Regulating Handling Raisin Administrative Committee § 989.30 Selection. The Secretary shall.... Such selections may be made from nominations certified pursuant to § 989.29 or from other eligible...

  17. Lasers à électrons libres de courtes longueurs d'onde: état de l'art et perspectives

    NASA Astrophysics Data System (ADS)

    Couprie, M. E.

    2003-06-01

    Les Lasers à Électrons Libres (LEL) sont des sources de rayonnement cohérent et accordable, basées sur l'interaction d'un faisceau d'électrons relativistes circulant dans le champ magnétique magnétique périodique et permanent créé par un “onduleur”. et d'une onde optique. L'onde lumineuse peut être issue de l'émission de rayonnement synchroton émise par le paquet d'électrons a chaque passage pour un système super-radiant en mode SASE (Self Amplified Spontaneous Emission- Emission Spontanée Auto-Amplifiée) ou issue du rayonnement synchrotron stocké dans une cavité optique, en mode oscillateur, ou d'une onde laser externe, en mode génération d'harmonique. Sous certaines conditions, l'onde lumineuse est amplifiée, au détriment de l'énergie cinétique des électrons, ce qui conduit à l'effet laser. L'état de l'art des sources Laser à Electrons Libres dans l'UV et le VUV est présenté, en indiquant les complémentarités résultant des différentes configurations tant du point de vue des sources que de leurs utilisations. Les projets de sources de courte longueur d'onde proposés sont ensuite discutés.

  18. 7 CFR 953.19 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 953.19 Section 953.19 Agriculture... STATES Order Regulating Handling Administrative Committee § 953.19 Selection. (a) The Secretary shall... § 953.18 and the selections shall be made pursuant to this section for the remainder of the 1968-69 term...

  19. Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

    PubMed Central

    Lim, Junghyun; Lachenmayer, M. Lenard; Wu, Shuai; Liu, Wenchao; Kundu, Mondira; Wang, Rong; Komatsu, Masaaki; Oh, Young J.; Zhao, Yanxiang; Yue, Zhenyu

    2015-01-01

    Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease. PMID:25723488

  20. 48 CFR 36.602-4 - Selection authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Selection authority. 36... Selection authority. (a) The final selection decision shall be made by the agency head or a designated selection authority. (b) The selection authority shall review the recommendations of the evaluation board...

  1. MicroRNA regulation of viral immunity, latency, and carcinogenesis of selected tumor viruses and HIV.

    PubMed

    Wang, Ling; Li, Guangyu; Yao, Zhi Q; Moorman, Jonathan P; Ning, Shunbin

    2015-09-01

    MicroRNAs (miRNAs) function as key regulators in immune responses and cancer development. In the contexts of infection with oncogenic viruses, miRNAs are engaged in viral persistence, latency establishment and maintenance, and oncogenesis. In this review, we summarize the potential roles and mechanisms of viral and cellular miRNAs in the host-pathogen interactions during infection with selected tumor viruses and HIV, which include (i) repressing viral replication and facilitating latency establishment by targeting viral transcripts, (ii) evading innate and adaptive immune responses via toll-like receptors, RIG-I-like receptors, T-cell receptor, and B-cell receptor pathways by targeting signaling molecules such as TRAF6, IRAK1, IKKε, and MyD88, as well as downstream targets including regulatory cytokines such as tumor necrosis factor α, interferon γ, interleukin 10, and transforming growth factor β, (iii) antagonizing intrinsic and extrinsic apoptosis pathways by targeting pro-apoptotic or anti-apoptotic gene transcripts such as the Bcl-2 family and caspase-3, (iv) modulating cell proliferation and survival through regulation of the Wnt, PI3K/Akt, Erk/MAPK, and Jak/STAT signaling pathways, as well as the signaling pathways triggered by viral oncoproteins such as Epstein-Barr Virus LMP1, by targeting Wnt-inhibiting factor 1, SHIP, pTEN, and SOCSs, and (v) regulating cell cycle progression by targeting cell cycle inhibitors such as p21/WAF1 and p27/KIP1. Further elucidation of the interaction between miRNAs and these key biological events will facilitate our understanding of the pathogenesis of viral latency and oncogenesis and may lead to the identification of miRNAs as novel targets for developing new therapeutic or preventive interventions. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Selective down-regulation of [(125)I]Y0-alpha-conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine.

    PubMed

    Mugnaini, M; Garzotti, M; Sartori, I; Pilla, M; Repeto, P; Heidbreder, C A; Tessari, M

    2006-01-01

    Prolonged exposure to nicotine, as occurs in smokers, results in up-regulation of all the neuronal nicotinic acetylcholine receptor subtypes studied so far, the only differences residing in the extent and time course of the up-regulation. alpha6beta2*-Nicotinic acetylcholine receptors are selectively enriched in the mesostriatal dopaminergic system and may play a crucial role in nicotine dependence. Here we show that chronic nicotine treatment (3mg/kg/day for two weeks, via s.c. osmotic minipumps) caused a significant decrease (36% on average) in the binding of [(125)I]Y(0)-alpha-conotoxin MII (a selective ligand for alpha6beta2*-nicotinic acetylcholine receptors in this system) to all the five regions of the rat dopaminergic pathway analyzed in this study. After one week of withdrawal, binding was still lower than control in striatal terminal regions (namely the caudate putamen and the accumbens shell and core). In somatodendritic regions (the ventral tegmental area and the substantia nigra) the decrease was significant at the end of the treatment and recovered within one day of withdrawal. This effect was not due to displacement of [(125)I]Y(0)-alpha-conotoxin MII binding by residual nicotine. In fact the binding was not changed by 565 ng/g nicotine (obtained with a single injection of nicotine), a concentration much higher than that found in the brain of rats chronically treated with nicotine (240 ng/g). In addition, consistent with previous studies reporting an up-regulation of other subtypes of nicotinic acetylcholine receptors, we found that nicotine exposure significantly increased (40% on average) the binding of [(125)I]epibatidine (a non-selective agonist at most neuronal heteromeric nicotinic acetylcholine receptors) in three up to five regions containing only alpha-conotoxin MII-insensitive [(125)I]epibatidine binding sites, namely the primary motor, somatosensory and auditory cortices. In conclusion, this work is the first to demonstrate that alpha6beta

  3. Targeted alpha therapy for cancer

    NASA Astrophysics Data System (ADS)

    Allen, Barry J.; Raja, Chand; Rizvi, Syed; Li, Yong; Tsui, Wendy; Zhang, David; Song, Emma; Qu, Chang Fa; Kearsley, John; Graham, Peter; Thompson, John

    2004-08-01

    Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The 213Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 µCi in human

  4. Strategies of organization and service for the critical-care laboratory.

    PubMed

    Fleisher, M; Schwartz, M K

    1990-08-01

    Critical-care medicine requires rapidity of treatment decisions and clinical management. To meet the objectives of critical-care medicine, the critical-care laboratory must consider four major aspects of laboratory organization in addition to analytical responsibilities: specimen collection and delivery, training of technologists, selection of reliable instrumentation, and efficient data dissemination. One must also consider the advantages and disadvantages of centralization vs decentralization, the influence of such a laboratory on patient care and personnel needs, and the space required for optimal operation. Centralization may lead to workflow interruption and increased turnaround time (TAT); decentralization requires redundancy of instrumentation and staff but may shorten TAT. Minimal TAT is the hallmark of efficient laboratory service. We surveyed 55 laboratories in 33 hospitals and found that virtually all hospitals with 200 or more beds had a critical-care laboratory operating as a satellite of the main laboratory. We present data on actual TAT, although these were available in only eight of the 15 routine laboratories that provided emergency service and in eight of the 40 critical-care laboratories. In meeting the challenges of an increasing workload, a reduced clinical laboratory work force, and the need to reduce TAT, changes in traditional laboratory practice are mandatory. An increased reliance on whole-blood analysis, for example, should eliminate delays associated with sample preparation, reduce the potential hazards associated with centrifugation, and eliminate excess specimen handling.

  5. 32 CFR 1602.21 - Selective Service Law.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Selective Service Law. 1602.21 Section 1602.21 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM DEFINITIONS § 1602.21 Selective Service Law. The term Selective Service Law includes the Military Selective Service Act...

  6. Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

    PubMed

    Rogers, M B

    1996-01-01

    The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

  7. Differential regulation of proximal and distal Vbeta segments upstream of a functional VDJbeta1 rearrangement upon beta-selection.

    PubMed

    Brady, Brenna L; Bassing, Craig H

    2011-09-15

    Developmental stage-specific regulation of transcriptional accessibility helps control V(D)J recombination. Vβ segments on unrearranged TCRβ alleles are accessible in CD4(-)/CD8(-) (double-negative [DN]) thymocytes, when they recombine, and inaccessible in CD4(+)/CD8(+) (double-positive [DP]) thymocytes, when they do not rearrange. Downregulation of Vβ accessibility on unrearranged alleles is linked with Lat-dependent β-selection signals that inhibit Vβ rearrangement, stimulate Ccnd3-driven proliferation, and promote DN-to-DP differentiation. Transcription and recombination of Vβs on VDJβ-rearranged alleles in DN cells has not been studied; Vβs upstream of functional VDJβ rearrangements have been found to remain accessible, yet not recombine, in DP cells. To elucidate contributions of β-selection signals in regulating Vβ transcription and recombination on VDJβ-rearranged alleles, we analyzed wild-type, Ccnd3(-/-), and Lat(-/-) mice containing a preassembled functional Vβ1DJCβ1 (Vβ1(NT)) gene. Vβ10 segments located just upstream of this VDJCβ1 gene were the predominant germline Vβs that rearranged in Vβ1(NT/NT) and Vβ1(NT/NT)Ccnd3(-/-) thymocytes, whereas Vβ4 and Vβ16 segments located further upstream rearranged at similar levels as Vβ10 in Vβ1(NT/NT)Lat(-/-) DN cells. We previously showed that Vβ4 and Vβ16, but not Vβ10, are transcribed on Vβ1(NT) alleles in DP thymocytes; we now demonstrate that Vβ4, Vβ16, and Vβ10 are transcribed at similar levels in Vβ1(NT/NT)Lat(-/-) DN cells. These observations indicate that suppression of Vβ rearrangements is not dependent on Ccnd3-driven proliferation, and DN residence can influence the repertoire of Vβs that recombine on alleles containing an assembled VDJCβ1 gene. Our findings also reveal that β-selection can differentially silence rearrangement of germline Vβ segments located proximal and distal to functional VDJβ genes.

  8. 48 CFR 436.602-4 - Selection authority.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Selection authority. 436... Selection authority. (a) The HCA shall serve as the selection authority in accordance with FAR 36.602-4. The... selection authority. (b) A copy of the final selection, inclusive of the supporting documents, shall be...

  9. 29 CFR 1605.3 - Selection practices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Selection practices. 1605.3 Section 1605.3 Labor Regulations Relating to Labor (Continued) EQUAL EMPLOYMENT OPPORTUNITY COMMISSION GUIDELINES ON DISCRIMINATION BECAUSE OF RELIGION § 1605.3 Selection practices. (a) Scheduling of tests or other selection procedures...

  10. Development and test of selected model pedestrian safety regulations

    DOT National Transportation Integrated Search

    1981-04-01

    Two model regulations to remove parking--one from suburban streets in daylight hours and one on the last 50 feet of the approach to crosswalks--were designed in previous work to prevent pedestrian dart and dash accidents by removing screening vehicle...

  11. 14 CFR 183.11 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Selection. 183.11 Section 183.11 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ADMINISTRATIVE REGULATIONS REPRESENTATIVES OF THE ADMINISTRATOR Certification of Representatives § 183.11 Selection. (a) The...

  12. Characterizing Age-Related Positivity Effects in Situation Selection

    ERIC Educational Resources Information Center

    Sands, Molly; Livingstone, Kimberly M.; Isaacowitz, Derek M.

    2018-01-01

    Situation selection--choosing to enter or avoid situations based on how they will likely make you feel--is theorized to be a useful emotion regulation strategy, especially in older age. However, research on the use of situation selection for emotion regulation is limited, and the existing findings about age differences are mixed, with some studies…

  13. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

    PubMed

    Chen, Jun; Joshi, Shailen K; DiDomenico, Stanley; Perner, Richard J; Mikusa, Joe P; Gauvin, Donna M; Segreti, Jason A; Han, Ping; Zhang, Xu-Feng; Niforatos, Wende; Bianchi, Bruce R; Baker, Scott J; Zhong, Chengmin; Simler, Gricelda H; McDonald, Heath A; Schmidt, Robert G; McGaraughty, Steve P; Chu, Katharine L; Faltynek, Connie R; Kort, Michael E; Reilly, Regina M; Kym, Philip R

    2011-05-01

    Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects. Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  14. Development of a qPCR Strategy to Select Bean Genes Involved in Plant Defense Response and Regulated by the Trichoderma velutinum - Rhizoctonia solani Interaction.

    PubMed

    Mayo, Sara; Cominelli, Eleonora; Sparvoli, Francesca; González-López, Oscar; Rodríguez-González, Alvaro; Gutiérrez, Santiago; Casquero, Pedro A

    2016-01-01

    Bean production is affected by a wide diversity of fungal pathogens, among them Rhizoctonia solani is one of the most important. A strategy to control bean infectious diseases, mainly those caused by fungi, is based on the use of biocontrol agents (BCAs) that can reduce the negative effects of plant pathogens and also can promote positive responses in the plant. Trichoderma is a fungal genus that is able to induce the expression of genes involved in plant defense response and also to promote plant growth, root development and nutrient uptake. In this article, a strategy that combines in silico analysis and real time PCR to detect additional bean defense-related genes, regulated by the presence of Trichoderma velutinum and/or R. solani has been applied. Based in this strategy, from the 48 bean genes initially analyzed, 14 were selected, and only WRKY33, CH5b and hGS showed an up-regulatory response in the presence of T. velutinum. The other genes were or not affected (OSM34) or down-regulated by the presence of this fungus. R. solani infection resulted in a down-regulation of most of the genes analyzed, except PR1, OSM34 and CNGC2 that were not affected, and the presence of both, T. velutinum and R. solani, up-regulates hGS and down-regulates all the other genes analyzed, except CH5b which was not significantly affected. As conclusion, the strategy described in the present work has been shown to be effective to detect genes involved in plant defense, which respond to the presence of a BCA or to a pathogen and also to the presence of both. The selected genes show significant homology with previously described plant defense genes and they are expressed in bean leaves of plants treated with T. velutinum and/or infected with R. solani.

  15. 45 CFR 1634.9 - Selection criteria.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Selection criteria. 1634.9 Section 1634.9 Public Welfare Regulations Relating to Public Welfare (Continued) LEGAL SERVICES CORPORATION COMPETITIVE BIDDING FOR GRANTS AND CONTRACTS § 1634.9 Selection criteria. (a) The criteria to be used to select among...

  16. 7 CFR 1216.42 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Selection. 1216.42 Section 1216.42 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... INFORMATION ORDER Peanut Promotion, Research, and Information Order National Peanut Board § 1216.42 Selection...

  17. Emotion regulation through listening to music in everyday situations.

    PubMed

    Thoma, Myriam V; Ryf, Stefan; Mohiyeddini, Changiz; Ehlert, Ulrike; Nater, Urs M

    2012-01-01

    Music is a stimulus capable of triggering an array of basic and complex emotions. We investigated whether and how individuals employ music to induce specific emotional states in everyday situations for the purpose of emotion regulation. Furthermore, we wanted to examine whether specific emotion-regulation styles influence music selection in specific situations. Participants indicated how likely it would be that they would want to listen to various pieces of music (which are known to elicit specific emotions) in various emotional situations. Data analyses by means of non-metric multidimensional scaling revealed a clear preference for pieces of music that were emotionally congruent with an emotional situation. In addition, we found that specific emotion-regulation styles might influence the selection of pieces of music characterised by specific emotions. Our findings demonstrate emotion-congruent music selection and highlight the important role of specific emotion-regulation styles in the selection of music in everyday situations.

  18. 32 CFR 1624.3 - Age selection groups.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Age selection groups. 1624.3 Section 1624.3 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM INDUCTIONS § 1624.3 Age selection groups. Age selection groups are established as follows: (a) The age 20 selection group for each calendar year consists of...

  19. 32 CFR 1624.3 - Age selection groups.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Age selection groups. 1624.3 Section 1624.3 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM INDUCTIONS § 1624.3 Age selection groups. Age selection groups are established as follows: (a) The age 20 selection group for each calendar year consists of...

  20. 32 CFR 1624.3 - Age selection groups.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Age selection groups. 1624.3 Section 1624.3 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM INDUCTIONS § 1624.3 Age selection groups. Age selection groups are established as follows: (a) The age 20 selection group for each calendar year consists of...

  1. 32 CFR 1624.3 - Age selection groups.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false Age selection groups. 1624.3 Section 1624.3 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM INDUCTIONS § 1624.3 Age selection groups. Age selection groups are established as follows: (a) The age 20 selection group for each calendar year consists of...

  2. 32 CFR 1624.3 - Age selection groups.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Age selection groups. 1624.3 Section 1624.3 National Defense Other Regulations Relating to National Defense SELECTIVE SERVICE SYSTEM INDUCTIONS § 1624.3 Age selection groups. Age selection groups are established as follows: (a) The age 20 selection group for each calendar year consists of...

  3. 48 CFR 715.370 - Alternative source selection procedures.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... selection procedures. 715.370 Section 715.370 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Source Selection 715.370 Alternative source selection procedures. The following selection procedures may be used, when...

  4. 7 CFR 1250.329 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Selection. 1250.329 Section 1250.329 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Research and Promotion Order Egg Board § 1250.329 Selection. From the nominations made pursuant to § 1250...

  5. 7 CFR 924.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 924.23 Section 924.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements....23 Selection. From the nominations made pursuant to § 924.22, or from other qualified persons, the...

  6. 5 CFR 317.603 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Selection. 317.603 Section 317.603 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT IN THE SENIOR EXECUTIVE SERVICE Noncareer and Limited Appointments § 317.603 Selection. An agency may make a noncareer or...

  7. The Fas/CD95 Receptor Regulates the Death of Autoreactive B Cells and the Selection of Antigen-Specific B Cells

    PubMed Central

    Koncz, Gabor; Hueber, Anne-Odile

    2012-01-01

    Cell death receptors have crucial roles in the regulation of immune responses. Here we review recent in vivo data confirming that the Fas death receptor (TNFSR6) on B cells is important for the regulation of autoimmunity since the impairment of only Fas function on B cells results in uncontrolled autoantibody production and autoimmunity. Fas plays a role in the elimination of the non-specific and autoreactive B cells in germinal center, while during the selection of antigen-specific B cells different escape signals ensure the resistance to Fas-mediated apoptosis. Antigen-specific survival such as BCR or MHCII signal or coreceptors (CD19) cooperating with BCR inhibits the formation of death inducing signaling complex. Antigen-specific survival can be reinforced by antigen-independent signals of IL-4 or CD40 overproducing the anti-apoptotic members of the Bcl-2 family proteins. PMID:22848207

  8. 7 CFR 920.23 - Selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Selection. 920.23 Section 920.23 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Administrative Body § 920.23 Selection. From the nominations made pursuant to § 920.22, or from other qualified...

  9. In Vivo, Fatty Acid Translocase (CD36) Critically Regulates Skeletal Muscle Fuel Selection, Exercise Performance, and Training-induced Adaptation of Fatty Acid Oxidation*

    PubMed Central

    McFarlan, Jay T.; Yoshida, Yuko; Jain, Swati S.; Han, Xioa-Xia; Snook, Laelie A.; Lally, James; Smith, Brennan K.; Glatz, Jan F. C.; Luiken, Joost J. F. P.; Sayer, Ryan A.; Tupling, A. Russell; Chabowski, Adrian; Holloway, Graham P.; Bonen, Arend

    2012-01-01

    For ∼40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (−21%) and oxidation (−25%), intramuscular lipids (less than or equal to −31%), and hepatic glycogen (−20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (−41%), oxidation (−37%), and exercise duration (−44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27–55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84–90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO. PMID:22584574

  10. Comments on the Lambert case: the rulings of the French Conseil d'État and the European Court of Human Rights.

    PubMed

    Veshi, Denard

    2017-06-01

    This study examines the decisions of the French Conseil d'Etat (Supreme Administrative Court) and the European Court of Human Rights in the Lambert case concerning the withdrawal of life-sustaining treatments. After presenting the facts of this case, the main legal question will be analyzed from an ethical and medical standpoint. The decisions of the Conseil d'État and then of the European Court of Human Rights are studied from a comparative legal perspective. This commentary focuses on the autonomous will of an unconscious patient and on the judicial interpretation of the right to life as recognized in article 2 of the European Convention on Human Rights. Furthermore, it medically classifies artificial nutrition and hydration (ANH) as a "treatment" which has ethical and legal implications. While the majority of the bioethical community considers ANH a medical treatment, a minority argues that ANH is basic care. This classification is ambiguous and has conflicting legal interpretations. In the conclusion, the author highlights how a French lawmaker in February 2016, finally clarified the status of ANH as a medical treatment which reconciled the different values at stake.

  11. Treatment with selective estrogen receptor modulators regulates myelin specific T-cells and suppresses experimental autoimmune encephalomyelitis.

    PubMed

    Bebo, Bruce F; Dehghani, Babak; Foster, Scott; Kurniawan, Astrid; Lopez, Francisco J; Sherman, Larry S

    2009-05-01

    Steroidal estrogens can regulate inflammatory immune responses and may be involved in the suppression of multiple sclerosis (MS) during pregnancy. However, the risks and side effects associated with steroidal estrogens may limit their usefulness for long-term MS therapy. Selective estrogen receptor modulators (SERMs) could provide an alternative therapeutic strategy, because they behave as estrogen agonists in some tissues, but are either inert or behave like estrogen antagonists in other tissues. In this study, we investigated the ability of two commercially available SERMs (tamoxifen and raloxifene) to regulate myelin specific immunity and experimental autoimmune encephalomyelitis (EAE) in mice. Both tamoxifen and raloxifene suppressed myelin antigen specific T-cell proliferation. However, tamoxifen was more effective in this regard. Tamoxifen treatment reduced the induction of major histocompatibility complex II by lipopolysaccharide stimulated dendritic cells and decreased their ability to activate myelin specific T-cells. At lower doses, tamoxifen was found to increase the levels of Th2 transcription factors and induce a Th2 bias in cultures of myelin-specific splenocytes. EAE symptoms and the degree of demyelination were less severe in mice treated with tamoxifen than in control mice. These findings support the notion that tamoxifen or related SERMs are potential agents that could be used in the treatment of inflammatory autoimmune disorders that affect the central nervous system.

  12. Reversible switch between the nanoporous and the nonporous state of amphiphilic block copolymer films regulated by selective swelling.

    PubMed

    Yan, Nina; Wang, Yong

    2015-09-21

    Switchable nanoporous films, which can repeatedly alternate their porosities, are of great interest in a diversity of fields. Currently these intelligent materials are mostly based on polyelectrolytes and their porosities can change only in relatively narrow ranges, typically under wet conditions, severely limiting their applications. Here we develop a new system, which is capable of reversibly switching between a highly porous state and a nonporous state dozens of times regulated simply by exposure to selective solvents. In this system nanopores are created or reversibly eliminated in films of a block copolymer, polystyrene-block-poly(2-vinyl pyridine) (PS-b-P2VP), by exposing the films to P2VP-selective or PS-selective solvents, respectively. The mechanism of the switch is based on the selective swelling of the constituent blocks in corresponding solvents, which is a nondestructive and easily controllable process enabling the repeatable and ample switch between the open and the closed state. Systematic microscopic and ellipsometric characterization methods are performed to elucidate the pore-closing course induced by nonsolvents and the cycling between the pore-open and the pore-closed state up to 20 times. The affinity of the solvent for PS blocks is found to play a dominating role in determining the pore-closing process and the porosities of the pore-open films increase with the cycling numbers as a result of loose packing conditions of the polymer chains. We finally demonstrate the potential applications of these films as intelligent antireflection coatings and drug carriers.

  13. Site-Selective Regulation of Platelet-Derived Growth Factor β Receptor Tyrosine Phosphorylation by T-Cell Protein Tyrosine Phosphatase

    PubMed Central

    Persson, Camilla; Sävenhed, Catrine; Bourdeau, Annie; Tremblay, Michel L.; Markova, Boyka; Böhmer, Frank D.; Haj, Fawaz G.; Neel, Benjamin G.; Elson, Ari; Heldin, Carl-Henrik; Rönnstrand, Lars; Östman, Arne; Hellberg, Carina

    2004-01-01

    The platelet-derived growth factor (PDGF) β receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF β receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF β receptor, we compared PDGF β receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF β receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cγ1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cγ1 activity and migratory hyperresponsiveness to PDGF. PDGF β receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPɛ ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF β receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors. PMID:14966296

  14. Multiple pre- and post-analytical lean approaches to the improvement of the laboratory turnaround time in a large core laboratory.

    PubMed

    Lou, Amy H; Elnenaei, Manal O; Sadek, Irene; Thompson, Shauna; Crocker, Bryan D; Nassar, Bassam A

    2017-10-01

    Core laboratory (CL), as a new business model, facilitates consolidation and integration of laboratory services to enhance efficiency and reduce costs. This study evaluates the impact of total laboratory automation system (TLA), electric track vehicle (ETV) system and auto-verification (AV) of results on overall turnaround time (TAT) (phlebotomy to reporting TAT: PR-TAT) within a CL setting. Mean, median and percentage of outlier (OP) for PR-TAT were compared for pre- and post-CL eras using five representative tests based on different request priorities. Comparison studies were also carried out on the intra-laboratory TAT (in-lab to reporting TAT: IR-TAT) and the delivery TAT (phlebotomy to in-lab TAT: PI-TAT) to reflect the efficiency of the TLA (both before and after introducing result AV) and ETV systems respectively. Median PR-TATs for the urgent samples were reduced on average by 16% across all representative analytes. Median PR-TATs for the routine samples were curtailed by 51%, 50%, 49%, 34% and 22% for urea, potassium, thyroid stimulating hormone (TSH), complete blood count (CBC) and prothrombin time (PT) respectively. The shorter PR-TAT was attributed to a significant reduction of IR-TAT through the TLA. However, the median PI-TAT was delayed when the ETV was used. Application of various AV rules shortened the median IR-TATs for potassium and urea. However, the OP of PR-TAT for the STAT requests exceeding 60min were all higher than those from the pre-CL era. TLA and auto-verification rules help to efficiently manage substantial volumes of urgent and routine samples. However, the ETV application as it stands shows a negative impact on the PR-TAT. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  15. CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    PubMed Central

    2012-01-01

    Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822–829 (2011)]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target. PMID

  16. Role of the Twin-Arginine Translocation Pathway in Staphylococcus▿ †

    PubMed Central

    Biswas, Lalitha; Biswas, Raja; Nerz, Christiane; Ohlsen, Knut; Schlag, Martin; Schäfer, Tina; Lamkemeyer, Tobias; Ziebandt, Anne-Kathrin; Hantke, Klaus; Rosenstein, Ralf; Götz, Friedrich

    2009-01-01

    In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB. PMID:19633084

  17. Structure-dependent water transport across nanopores of carbon nanotubes: toward selective gating upon temperature regulation.

    PubMed

    Zhao, Kuiwen; Wu, Huiying

    2015-04-28

    Determining water structure in nanopores and its influence on water transport behaviour is of great importance for understanding and regulating the transport across nanopores. Here we report an ultrafast-slow flow transition phenomenon for water transport across nanopores of carbon nanotubes owing to the change in water structure in nanopores induced by temperature. By performing extensive molecular dynamics simulations, we show the dependence of water transport behaviours on water structures. Our results indicate that owing to the change in water structure in nanopores, water flux across nanopores with certain pore sizes decreases sharply (nearly 3 orders of magnitude) with the decreasing temperature. This phenomenon is very sensitive to the pore size. The threshold temperatures for the occurrence of the ultrafast-slow flow transition for water transport are also determined for various pore sizes. These findings suggest a novel protocol for selective gating of water and proton conduction across nanopores and temperature-controlled drug release.

  18. 48 CFR 36.301 - Use of two-phase design-build selection procedures.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Use of two-phase design-build selection procedures. 36.301 Section 36.301 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION SPECIAL CATEGORIES OF CONTRACTING CONSTRUCTION AND ARCHITECT-ENGINEER CONTRACTS Two-Phase Design-Build Selection Procedures 36.301...

  19. 7 CFR 2201.18 - Application selection.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Application selection. 2201.18 Section 2201.18 Agriculture Regulations of the Department of Agriculture (Continued) LOCAL TELEVISION LOAN GUARANTEE BOARD... selection. (a) Application Priority. When evaluating applications to determine which Project or combinations...

  20. Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

    PubMed Central

    Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

    1989-01-01

    The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877