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

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

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

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.

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

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.

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.

Full trans-activation mediated by the immediate-early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence.

PubMed

Kim, Seong K; Shakya, Akhalesh K; O'Callaghan, Dennis J

2016-01-04

The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt -89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS). Copyright © 2015 Elsevier B.V. All rights reserved.

EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

PubMed

Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

2014-05-09

Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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.

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.

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.

All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation.

PubMed

Fontán-Gabás, Lorena; Oliemuller, Erik; Martínez-Irujo, Juan José; de Miguel, Carlos; Rouzaut, Ana

2007-01-01

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.

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

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

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.

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

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

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

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

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

Identifying cis-mediators for trans-eQTLs across many human tissues using genomic mediation analysis.

PubMed

Yang, Fan; Wang, Jiebiao; Pierce, Brandon L; Chen, Lin S

2017-11-01

The impact of inherited genetic variation on gene expression in humans is well-established. The majority of known expression quantitative trait loci (eQTLs) impact expression of local genes ( cis -eQTLs). More research is needed to identify effects of genetic variation on distant genes ( trans -eQTLs) and understand their biological mechanisms. One common trans -eQTLs mechanism is "mediation" by a local ( cis ) transcript. Thus, mediation analysis can be applied to genome-wide SNP and expression data in order to identify transcripts that are " cis -mediators" of trans -eQTLs, including those " cis -hubs" involved in regulation of many trans -genes. Identifying such mediators helps us understand regulatory networks and suggests biological mechanisms underlying trans -eQTLs, both of which are relevant for understanding susceptibility to complex diseases. The multitissue expression data from the Genotype-Tissue Expression (GTEx) program provides a unique opportunity to study cis -mediation across human tissue types. However, the presence of complex hidden confounding effects in biological systems can make mediation analyses challenging and prone to confounding bias, particularly when conducted among diverse samples. To address this problem, we propose a new method: Genomic Mediation analysis with Adaptive Confounding adjustment (GMAC). It enables the search of a very large pool of variables, and adaptively selects potential confounding variables for each mediation test. Analyses of simulated data and GTEx data demonstrate that the adaptive selection of confounders by GMAC improves the power and precision of mediation analysis. Application of GMAC to GTEx data provides new insights into the observed patterns of cis -hubs and trans -eQTL regulation across tissue types. © 2017 Yang et al.; Published by Cold Spring Harbor Laboratory Press.

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.

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.

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.

Identifying cis-mediators for trans-eQTLs across many human tissues using genomic mediation analysis

PubMed Central

Yang, Fan; Wang, Jiebiao; Pierce, Brandon L.; Chen, Lin S.

2017-01-01

The impact of inherited genetic variation on gene expression in humans is well-established. The majority of known expression quantitative trait loci (eQTLs) impact expression of local genes (cis-eQTLs). More research is needed to identify effects of genetic variation on distant genes (trans-eQTLs) and understand their biological mechanisms. One common trans-eQTLs mechanism is “mediation” by a local (cis) transcript. Thus, mediation analysis can be applied to genome-wide SNP and expression data in order to identify transcripts that are “cis-mediators” of trans-eQTLs, including those “cis-hubs” involved in regulation of many trans-genes. Identifying such mediators helps us understand regulatory networks and suggests biological mechanisms underlying trans-eQTLs, both of which are relevant for understanding susceptibility to complex diseases. The multitissue expression data from the Genotype-Tissue Expression (GTEx) program provides a unique opportunity to study cis-mediation across human tissue types. However, the presence of complex hidden confounding effects in biological systems can make mediation analyses challenging and prone to confounding bias, particularly when conducted among diverse samples. To address this problem, we propose a new method: Genomic Mediation analysis with Adaptive Confounding adjustment (GMAC). It enables the search of a very large pool of variables, and adaptively selects potential confounding variables for each mediation test. Analyses of simulated data and GTEx data demonstrate that the adaptive selection of confounders by GMAC improves the power and precision of mediation analysis. Application of GMAC to GTEx data provides new insights into the observed patterns of cis-hubs and trans-eQTL regulation across tissue types. PMID:29021290

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

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

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

Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

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

Kenney, S.; Kamine, J.; Markovitz, D.

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBVmore » gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.« less

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.

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

Mediation Analysis Demonstrates That Trans-eQTLs Are Often Explained by Cis-Mediation: A Genome-Wide Analysis among 1,800 South Asians

PubMed Central

Pierce, Brandon L.; Tong, Lin; Chen, Lin S.; Rahaman, Ronald; Argos, Maria; Jasmine, Farzana; Roy, Shantanu; Paul-Brutus, Rachelle; Westra, Harm-Jan; Franke, Lude; Esko, Tonu; Zaman, Rakibuz; Islam, Tariqul; Rahman, Mahfuzar; Baron, John A.; Kibriya, Muhammad G.; Ahsan, Habibul

2014-01-01

A large fraction of human genes are regulated by genetic variation near the transcribed sequence (cis-eQTL, expression quantitative trait locus), and many cis-eQTLs have implications for human disease. Less is known regarding the effects of genetic variation on expression of distant genes (trans-eQTLs) and their biological mechanisms. In this work, we use genome-wide data on SNPs and array-based expression measures from mononuclear cells obtained from a population-based cohort of 1,799 Bangladeshi individuals to characterize cis- and trans-eQTLs and determine if observed trans-eQTL associations are mediated by expression of transcripts in cis with the SNPs showing trans-association, using Sobel tests of mediation. We observed 434 independent trans-eQTL associations at a false-discovery rate of 0.05, and 189 of these trans-eQTLs were also cis-eQTLs (enrichment P<0.0001). Among these 189 trans-eQTL associations, 39 were significantly attenuated after adjusting for a cis-mediator based on Sobel P<10-5. We attempted to replicate 21 of these mediation signals in two European cohorts, and while only 7 trans-eQTL associations were present in one or both cohorts, 6 showed evidence of cis-mediation. Analyses of simulated data show that complete mediation will be observed as partial mediation in the presence of mediator measurement error or imperfect LD between measured and causal variants. Our data demonstrates that trans-associations can become significantly stronger or switch directions after adjusting for a potential mediator. Using simulated data, we demonstrate that this phenomenon is expected in the presence of strong cis-trans confounding and when the measured cis-transcript is correlated with the true (unmeasured) mediator. In conclusion, by applying mediation analysis to eQTL data, we show that a substantial fraction of observed trans-eQTL associations can be explained by cis-mediation. Future studies should focus on understanding the mechanisms underlying

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed Central

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-01-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses. Images PMID:2830625

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-03-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

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

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.

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.

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

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.

Enhancer Activation Requires Trans-Recruitment of a Mega Transcription Factor Complex

PubMed Central

Liu, Zhijie; Merkurjev, Daria; Yang, Feng; Li, Wenbo; Oh, Soohwan; Friedman, Meyer J.; Song, Xiaoyuan; Zhang, Feng; Ma, Qi; Ohgi, Kenneth; Krones, Anna; Rosenfeld, Michael G.

2014-01-01

Summary Enhancers provide critical information directing cell-type specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome. PMID:25303530

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

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.

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.

EHV-1 EICP22 protein sequences that mediate its physical interaction with the immediate-early protein are not sufficient to enhance the trans-activation activity of the IE protein.

PubMed

Derbigny, Wilbert A; Kim, Seong K; Jang, Hyung K; O'Callaghan, Dennis J

2002-03-20

The early 293 amino acid EICP22 protein (EICP22P) of equine herpesvirus 1 localizes within the nucleus and functions as an accessory regulatory protein (J. Virol. 68 (1994) 4329). Transient transfection assays indicated that although the EICP22P by itself only minimally trans-activates EHV-1 promoters, the EICP22P functions synergistically with the immediate-early protein (IEP) to enhance expression of EHV-1 early genes (J. Virol. 71 (1997) 1004). We previously showed that the EICP22 protein enhances the DNA-binding activity of the EHV-1 IEP and that it also physically interacts with the IEP (J. Virol. 74 (2000) 1425). In this communication, we employed transient trans-activation assays utilizing EICP22P deletion mutants to address whether the sequences required for EICP22P-IEP physical interactions are essential for EICP22P's ability to interact synergistically with the IEP. Assays employing various classes of the EHV-1 promoters fused to the chloramphenicol acetyl-transferase (CAT) reporter gene indicated that: (1) neither full length nor any of the EICP22P mutants tested was able to overcome repression of the IE promoter elicited by the IEP, (2) the full-length EICP22P interacted synergistically with the IEP to trans-activate the early and late promoters tested, and (3) all of the EICP22P mutants, including those that were able to physically interact with IEP and itself, failed to function synergistically with the IEP to trans-activate representative EHV-1 early and late promoters. The results suggest that EICP22P sequences required for its interaction with the IE protein are not sufficient to mediate its synergistic effect on the trans-activation function of the IEP. The possible explanations as to why sequences in addition to those that mediate EICP22P-IEP interaction and EICP22P self-interactions are essential for the synergistic function of EICP22P are discussed.

Small RNA-Mediated trans-Nuclear and trans-Element Communications in Tetrahymena DNA Elimination.

PubMed

Noto, Tomoko; Mochizuki, Kazufumi

2018-06-18

Epigenetic inheritance of acquired traits is widespread among eukaryotes, but how and to what extent such information is transgenerationally inherited is still unclear. The patterns of programmed DNA elimination in ciliates are epigenetically and transgenerationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence and role of such small-RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is, indeed, determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small-RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small-RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

[Preliminary study on transdermal characteristics and sunface anesthetic effects of lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome in animals].

PubMed

Wang, Yue; Zhang, Lianyun; Li, Changyi; Wang, Hanjie; Li, Qin

2015-07-01

To prepare a new dental topical anesthetics, lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome (LID-TAT-N), and to evaluate its transdermal properties and topical anesthesia effects. LID-TAT-N was prepared using reverse-phase evaporation method, and lidocaine loaded conventional liposome (LID-CL) was prepared in the same manner as positive control. The diameter, ζ potential and encapsulation efficiency of LID-TAT-N and LID-CL were measured. The skin permeation of LID-TAT-N was examined, and compared with LID-CL and lidocaine injection (LID-IJ, as negative control), using a Franz diffusion cell mounted with depilated mouse skin in vitro for 12 hours. Each experiment was repeated six times. The anesthetic effect of the new topical anesthetic was investigated on the cornea of rabbits. The mean diameter of LID-TAT-N was smaller than that of LID-CL [(152.7 ± 10.6) nm vs. (259.5 ± 15.5) nm, P < 0.01]. The 12 h cumulative permeation amount was significantly higher in LID-TAT-N group [(1 340.0 ± 97.5) µg · cm(-2)] than those of LID-CL and LID-IJ groups [(1 060.6 ± 80.2), (282.6 ± 65.1) µg · cm(-2), respectively, P < 0.05]. Rabbit corneal reflex results showed that LID-TAT-N had anesthetic effect and the duration of analgesia [(24.8 ± 2.8) min] was also longer than that of LID-IJ [(14.5 ± 2.3) min, P < 0.05]. LID-TAT-N had good transdermal ability, and the advanced skin penetration feature can improve its tropical anesthetic effect.

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.

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.

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

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

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

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

Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans -activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a -tdTomato- or Drd2 -eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1. SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury

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.

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

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

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

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.

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.

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.

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.

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.

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

A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

PubMed

Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

2013-04-01

This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

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

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.

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

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.

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.

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.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the

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

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

PubMed

Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

2010-03-01

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

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.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

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.

Repair Activity of trans-Resveratrol toward 2'-Deoxyguanosine Radicals.

PubMed

Cheng, Xing; An, Ping; Li, Shujin; Zhou, Liping

2018-04-26

In the present study, the repair activity of trans-resveratrol toward 2'-deoxyguanosine (dGuo) radicals in polar and nonpolar solvents was studied using density functional theory. The hydrogen transfer/proton coupled electron transfer and single electron transfer (SET) mechanisms between trans-resveratrol and dGuo-radicals were considered. Taking into consideration the molar fraction of neutral trans-resveratrol (ROH) and anionic trans-resveratrol (RO - ), the overall rate constants for repairing dGuo-radicals by trans-resveratrol are 9.94 × 10 8 and 2.01 × 10 9 dm 3 mol -1 s -1 in polar and nonpolar solvents, respectively, and the overall rate constant of repairing cation radical (dGuo •+ ) by trans-resveratrol via an SET mechanism is 7.17 × 10 9 dm 3 mol -1 s -1 . The repair activity of RO - toward dGuo-radicals is better than that of ROH, but the repair activity of ROH toward dGuo •+ is better than that of RO - . Unfortunately, neither ROH nor RO - can repair the 2'-deoxyribose radicals of dGuo. It can therefore be concluded that trans-resveratrol is an effective antioxidant for repairing base radicals of dGuo and dGuo •+ . The study can help us understand the repair activity of trans-resveratrol toward dGuo radicals.

Full trans–activation mediated by the immediate–early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence

PubMed Central

Kim, Seong K.; Shakya, Akhalesh K.; O'Callaghan, Dennis J.

2015-01-01

The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt −89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS). PMID:26541315

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.

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.

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

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.

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

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.

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

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

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

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…

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.

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.

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.

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

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.

trans-2-Tritylcyclohexanol as a chiral auxiliary in permanganate-mediated oxidative cyclization of 2-methylenehept-5-enoates: application to the synthesis of trans-(+)-linalool oxide.

PubMed

Al Hazmi, Ali M; Sheikh, Nadeem S; Bataille, Carole J R; Al-Hadedi, Azzam A M; Watkin, Sam V; Luker, Tim J; Camp, Nicholas P; Brown, Richard C D

2014-10-03

The permanganate-mediated oxidative cyclization of a series of 2-methylenehept-5-eneoates bearing different chiral auxiliaries was investigated, leading to the discovery of trans-2-tritylcyclohexanol (TTC) as a highly effective chiral controller for the formation of the 2,5-substituted THF diol product with high diastereoselectivity (dr ∼97:3). Chiral resolution of (±)-TTC, prepared in one step from cyclohexene oxide, afforded (-)-(1S,2R)-TTC (er >99:1), which was applied to the synthesis of (+)-trans-(2S,5S)-linalool oxide.

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

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.

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

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

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.

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

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.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach.

PubMed

Poddar, Sushmita; Loh, Pei She; Ooi, Zi Hao; Osman, Farhana; Eul, Joachim; Patzel, Volker

2018-06-01

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3' exon replacement (3'ER), 5' exon replacement (5'ER) correlated with the thermodynamic stability of the tsRNA 3' end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Powdered activated carbon adsorption of two fishy odorants in water: Trans,trans-2,4-heptadienal and trans,trans-2,4-decadienal.

PubMed

Li, Xin; Wang, Jun; Zhang, Xiaojian; Chen, Chao

2015-06-01

Powdered activated carbon (PAC) adsorption of two fishy odorants, trans,trans-2,4-heptadienal (HDE) and trans,trans-2,4-decadienal (DDE), was investigated. Both the pseudo first-order and the pseudo second-order kinetic models well described the kinetics curves, and DDE was more readily removed by PAC. In isotherm tests, both Freundlich and Modified Freundlich isotherms fitted the experimental data well. PAC exhibited a higher adsorption capacity for DDE than for HDE, which could be ascribed to the difference in their hydrophobicity. The calculated thermodynamic parameters (ΔG0, ΔH0, and ΔS0) indicated an exothermic and spontaneous adsorption process. PAC dosage, pH, and natural organic matter (NOM) presence were found to influence the adsorption process. With increasing PAC dosage, the pseudo first-order and pseudo second-order rate constants both increased. The value of pH had little influence on HDE or DDE molecules but altered the surface charge of PAC, and the maximum adsorption capacity occurred at pH9. The presence of NOM, especially the fraction with molecular weight less than 1k Dalton, hindered the adsorption. The study showed that preloaded NOM impaired the adsorption capacity of HDE or DDE more severely than simultaneously fed NOM did. Copyright © 2015. Published by Elsevier B.V.

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.

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.

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.

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.

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.

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.

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

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.

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

Probing interaction of a fluorescent ligand with HIV TAR RNA

NASA Astrophysics Data System (ADS)

Qi, Liang; Zhang, Jing; He, Tian; Huo, Yuan; Zhang, Zhi-Qi

2017-02-01

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

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.

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

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.

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.

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

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.

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

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

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.

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

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.

NF-κB Activation in Hypothalamic Pro-opiomelanocortin Neurons Is Essential in Illness- and Leptin-induced Anorexia*

PubMed Central

Jang, Pil-Geum; Namkoong, Cherl; Kang, Gil Myoung; Hur, Man-Wook; Kim, Seung-Whan; Kim, Geun Hyang; Kang, Yeoungsup; Jeon, Min-Jae; Kim, Eun Hee; Lee, Myung-Shik; Karin, Michael; Baik, Ja-Hyun; Park, Joong-Yeol; Lee, Ki-Up; Kim, Young-Bum; Kim, Min-Seon

2010-01-01

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-κB (NF-κB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-κB. In vitro, NF-κB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-κB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-κB and melanocortin. Furthermore, disruption of IκB kinase-β, an upstream kinase of NF-κB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-κB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-κB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-κB also serves as a downstream signaling pathway of leptin. PMID:20097762

NF-kappaB activation in hypothalamic pro-opiomelanocortin neurons is essential in illness- and leptin-induced anorexia.

PubMed

Jang, Pil-Geum; Namkoong, Cherl; Kang, Gil Myoung; Hur, Man-Wook; Kim, Seung-Whan; Kim, Geun Hyang; Kang, Yeoungsup; Jeon, Min-Jae; Kim, Eun Hee; Lee, Myung-Shik; Karin, Michael; Baik, Ja-Hyun; Park, Joong-Yeol; Lee, Ki-Up; Kim, Young-Bum; Kim, Min-Seon

2010-03-26

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-kappaB (NF-kappaB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-kappaB. In vitro, NF-kappaB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-kappaB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-kappaB and melanocortin. Furthermore, disruption of I kappaB kinase-beta, an upstream kinase of NF-kappaB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-kappaB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-kappaB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-kappaB also serves as a downstream signaling pathway of leptin.

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.

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

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

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.

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

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

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

Tiam–Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion

PubMed Central

2016-01-01

Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph–ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB+ cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB+ cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion. PMID:27597758

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.

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.

Structural basis for human PECAM-1-mediated trans-homophilic cell adhesion

DOE PAGES

Hu, Menglong; Zhang, Hongmin; Liu, Qun; ...

2016-12-13

Cell adhesion involved in signal transduction, tissue integrity and pathogen infection is mainly mediated by cell adhesion molecules (CAM). One CAM member, platelet–endothelial-cell adhesion molecule-1 (PECAM-1), plays an important role in tight junction among endothelia cells, leukocyte trafficking, and immune response through its homophilic and heterophilic binding patterns. Both kinds of interactions, which lead to endogenous and exogenous signal transmission, are derived from extracellular immunoglobulin-like (IgL) domains and cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of PECAM-1. To date, the mechanism of trans-homophilic interaction of PECAM-1 remains unclear. Here, we present the crystal structure of PECAM-1 IgL1-2 trans-homo dimer. Both IgLmore » 1 and 2 adopt the classical Ig domain conformation comprised of two layers of β-sheets possessing antiparallel β-strands with each being anchored by a pair of cysteines forming a disulfide bond. The dimer interface includes hydrophobic and hydrophilic interactions. The Small-Angle X-ray Scattering (SAXS) envelope of PECAM-1 IgL1-6 supported such a dimer formation in solution. As a result, cell adhesion assays on wildtype and mutant PECAM-1 further characterized the structural determinants in cell junction and communication.« less

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking.

PubMed

Higgins, Chelsea D; Koellhoffer, Jayne F; Chandran, Kartik; Lai, Jonathan R

2013-10-01

We previously described potent inhibition of Ebola virus entry by a 'C-peptide' based on the GP2 C-heptad repeat region (CHR) targeted to endosomes ('Tat-Ebo'). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain-side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (~10(3)-fold reduction in infection at 40 μM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain-side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain-side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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

Dabney-Smith, Carole

system mechanism in chloroplasts will lead to a better understanding of the biogenesis of photosynthetic membranes potentially providing a means to engineer photosynthetic complexes into synthetic membranes for energy production. We are especially well prepared to undertake this project because we have developed a novel functional replacement assay, which was used to demonstrate a correlation of Tha4 oligomerization to transport. Thylakoids of plant chloroplasts provide a very robust, reliable assay to gain mechanistic detail about cpTat systems, providing most of the biochemical analyses to date. We plan to test our central hypothesis and accomplish the overall objective of this proposal by (1) Identifying the cpTat component(s) that interact with the mature domain of precursor during transport, (2) Determining the organization of the cpTat translocon, and (3) Comparing Tha4 topology in thylakoids during active transport and at rest. The proposed studies are innovative due to our ability to correlate structural changes in cpTat protein complexes during the transport of precursor. At the completion of this project, we expect to know the cpTat component(s) that interacts directly with the mature domain of the precursor, important because it is not known which components comprise the pore for passage of the mature domain. We also expect to know the arrangement of the components in the cpTat transport complex through direct interaction between Tha4 and the other CpTat components, a key point to establishing the mechanism of translocation. Lastly, we expect to correlate topological changes of Tha4 with precursor transport, key to establishing Tha4's role in the transport process. The successful completion of these studies is expected to have an important impact in understanding chloroplast biogenesis and assembly of photosynthetic complexes in plants and photosynthetic bacteria.« less

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.

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.

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.

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

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.

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.

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.

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

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.

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.

Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1.

PubMed

Leu, W M; Chen, L Y; Liaw, L L; Lee, Y H

1992-10-05

The tyrosinase of Streptomyces antibioticus is encoded by the second open reading frame, melC2 of the melanin operon (melC). The upstream open reading frame melC1 specifies a 146-amino acid protein with a typical NH2-terminal signal-peptide characteristic of a secretory protein. The MelC1 protein is involved in the transfer of copper ion to apotyrosinase MelC2 via binary complex formation (Lee, Y.-H. W., Chen, B.-F., Wu, S.-Y., Leu, W.-M., Lin, J.-J., Chen, C. W., and Lo, S. J. (1988) Gene (Amst.) 65, 71-81; Chen, L.-Y., Leu, W.-M., Wang, K.-T., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 20100-20107). To investigate whether the export of tyrosinase is also dependent on MelC1, a mutational study of its signal-peptide sequence was performed. Four different mutants were obtained. Mutation at the positively charged region (mutant M-6LE, Arg6-Arg7----Leu6-Glu7) or the hydrophobic region (mutant M-16D, Val16----Asp16) led to Mel- phenotypes. These lesions caused a severe 7-10-fold reduction of the export of both the MelC1 and MelC2 proteins and a concomitant accumulation of the two proteins in the cytosolic fraction. The cell-associated tyrosinase activity in M-6LE but not in the M-16D mutant was dramatically reduced to 4% of the activity found in the wild type strain, suggesting that the basic NH2 terminus of MelC1 is also important for the trans-activation function of this protein. Nevertheless, the defects on the trans-activation and/or secretory functions of MelC1 in mutants M-6LE and M-16D are not due to the impairment of the formation of the MelC1.MelC2 complex. The translation of melanin operon genes in these two mutants also decreased. In contrast, the tyrosinase activity and the secretion of MelC2 were not affected if the mutations occurred at the putative cleavage site of the signal peptidase (e.g. mutant M-29SM, Arg29-Ala30----Ser29-Met30 or mutant 29-SMG, Arg29-Ala30-Asp31----Ser29-Med30-Gly31+ ++). Additionally, tyrosinase activity and its export were

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

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

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.

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

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

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.

Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

PubMed

Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Anticoli, Simona; Manfredi, Francesco; Ferrantelli, Flavia; d'Ettorre, Gabriella; Schietroma, Ivan; Andreotti, Mauro; Federico, Maurizio

2017-09-01

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4 + T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4 + T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4 + T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4 + T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4 + T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4 + T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

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

USE OF TRANS-CONTEXTUAL MODEL-BASED PHYSICAL ACTIVITY COURSE IN DEVELOPING LEISURE-TIME PHYSICAL ACTIVITY BEHAVIOR OF UNIVERSITY STUDENTS.

PubMed

Müftüler, Mine; İnce, Mustafa Levent

2015-08-01

This study examined how a physical activity course based on the Trans-Contextual Model affected the variables of perceived autonomy support, autonomous motivation, determinants of leisure-time physical activity behavior, basic psychological needs satisfaction, and leisure-time physical activity behaviors. The participants were 70 Turkish university students (M age=23.3 yr., SD=3.2). A pre-test-post-test control group design was constructed. Initially, the participants were randomly assigned into an experimental (n=35) and a control (n=35) group. The experimental group followed a 12 wk. trans-contextual model-based intervention. The participants were pre- and post-tested in terms of Trans-Contextual Model constructs and of self-reported leisure-time physical activity behaviors. Multivariate analyses showed significant increases over the 12 wk. period for perceived autonomy support from instructor and peers, autonomous motivation in leisure-time physical activity setting, positive intention and perceived behavioral control over leisure-time physical activity behavior, more fulfillment of psychological needs, and more engagement in leisure-time physical activity behavior in the experimental group. These results indicated that the intervention was effective in developing leisure-time physical activity and indicated that the Trans-Contextual Model is a useful way to conceptualize these relationships.

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.

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

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.

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

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.

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.

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

Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons.

PubMed

Allette, Yohance M; Kim, Youngsook; Randolph, Aaron L; Smith, Jared A; Ripsch, Matthew S; White, Fletcher A

2017-06-16

Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent.

Suicide risk in trans populations: An application of minority stress theory.

PubMed

Tebbe, Elliot A; Moradi, Bonnie

2016-10-01

Drawing on minority stress theory, the present study tested the relations of minority stressors (i.e., experiences of prejudice and discrimination, internalized antitrans attitudes, fear of antitrans stigma), social support (i.e., friend, family, and significant other support), and substance use (i.e., drug and alcohol use) with depression and suicide risk in a sample of trans individuals. Depression was examined as a mediator of the relations of minority stressors and social support with suicide risk; drug and alcohol use were examined as direct correlates of suicide risk. Participants were 335 trans-identified individuals, diverse in gender identities (e.g., trans men, trans women, nonbinary gender identities). They were recruited using online social networks and they completed the study survey online via Qualtrics. Structural equation modeling was used to test hypothesized relations. Depression fully mediated the relations of perceived experiences of discrimination, fear of antitrans stigma, and friend support with suicide risk, and partially mediated the relation of internalized antitrans attitudes with suicide. Drug use was positively associated with suicide risk, whereas alcohol use was not linked with suicide risk. Exploratory comparisons across gender subgroups suggested that the pattern of relations among study variables was consistent across trans men, trans women, and individuals with nonbinary gender identities. These findings point to minority stressors, friend support, and drug use as potentially fruitful targets of prevention and intervention efforts to reduce depression and suicide risk in trans populations. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

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.

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction.

PubMed Central

Ikawa, Y; Shiraishi, H; Inoue, T

1999-01-01

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons. PMID:10075996

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.

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.

ILK mediates LPS-induced vascular adhesion receptor expression and subsequent leucocyte trans-endothelial migration.

PubMed

Hortelano, Sonsoles; López-Fontal, Raquel; Través, Paqui G; Villa, Natividad; Grashoff, Carsten; Boscá, Lisardo; Luque, Alfonso

2010-05-01

The inflammatory response to injurious agents is tightly regulated to avoid adverse consequences of inappropriate leucocyte accumulation or failed resolution. Lipopolysaccharide (LPS)-activated endothelium recruits leucocytes to the inflamed tissue through controlled expression of membrane-associated adhesion molecules. LPS responses in macrophages are known to be regulated by integrin-linked kinase (ILK); in this study, we investigated the role of ILK in the regulation of the LPS-elicited inflammatory response in endothelium. This study was performed on immortalized mouse endothelial cells (EC) isolated from lung and coronary vasculature. Cells were thoroughly characterized and the role of ILK in the regulation of the LPS response was investigated by suppressing ILK expression using siRNA and shRNA technologies. Phenotypic and functional analyses confirmed that the immortalized cells behaved as true EC. LPS induced the expression of the inflammatory genes E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). ILK knockdown impaired LPS-mediated endothelial activation by preventing the induction of ICAM-1 and VCAM-1. Blockade of the LPS-induced response inhibited the inflammatory-related processes of firm adhesion and trans-endothelial migration of leucocytes. ILK is involved in the expression of cell adhesion molecules by EC activated with the inflammatory stimulus LPS. This reduced expression modulates leucocyte adhesion to the endothelium and the extravasation process. This finding suggests ILK as a potential anti-inflammatory target for the development of vascular-specific treatments for inflammation-related diseases.

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

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

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

Distinct nuclear body components, PML and SMRT, regulate the trans-acting function of HTLV-1 Tax oncoprotein.

PubMed

Ariumi, Yasuo; Ego, Takeshi; Kaida, Atsushi; Matsumoto, Mikiko; Pandolfi, Pier Paolo; Shimotohno, Kunitada

2003-03-20

Several viruses target cellular promyelocytic leukemia (PML)-nuclear bodies (PML-NBs) to induce their disruption, marked morphological changes in these structures or the relocation to PML-NB components to the cytoplasm of infected cells. PML conversely interferes with viral replication. We demonstrate that PML acts as a coactivator for the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein without direct binding. Tax was identified within interchromatin granule clusters (IGCs)/RNA splicing bodies (SBs), not PML-NBs; Tax expression did not affect PML-NB formation. Moreover, PML and CBP/p300 cooperatively activated Tax-mediated HTLV-1-LTR-dependent gene expression. Interestingly, two PML mutants, PML-RAR and PMLDelta216-331, which fail to form PML-NBs, could also coactivate Tax-mediated trans-acting function but had no effect on retinoic acid receptor (RAR)- or p53-dependent gene expression. In contrast, SMRT (silencing mediator for retinoic acid and thyroid hormone receptors), a nuclear corepressor found within the matrix-associated deacetylase (MAD) nuclear body, relocalized into Tax-associated nuclear bodies upon coexpression with Tax. SMRT coactivated the trans-acting function of Tax through direct binding. Coexpression of SMRT and PML resulted in an additive activation of Tax trans-acting function. Thus, crosstalk between distinct nuclear bodies may control Tax function.

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.

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.

All-trans retinoic acid regulates hepatic bile acid homeostasis

PubMed Central

Yang, Fan; He, Yuqi; Liu, Hui-Xin; Tsuei, Jessica; Jiang, Xiaoyue; Yang, Li; Wang, Zheng-Tao; Wan, Yu-Jui Yvonne

2014-01-01

Retinoic acid (RA) and bile acids share common roles in regulating lipid homeostasis and insulin sensitivity. In addition, the receptor for RA (retinoid x receptor) is a permissive partner of the receptor for bile acids, farnesoid x receptor (FXR/NR1H4). Thus, RA can activate the FXR-mediated pathway as well. The current study was designed to understand the effect of all-trans RA on bile acid homeostasis. Mice were fed an all-trans RA-supplemented diet and the expression of 46 genes that participate in regulating bile acid homeostasis was studied. The data showed that all-trans RA has a profound effect in regulating genes involved in synthesis and transport of bile acids. All-trans RA treatment reduced the gene expression levels of Cyp7a1, Cyp8b1, and Akr1d1, which are involved in bile acid synthesis. All-trans RA also decreased the hepatic mRNA levels of Lrh-1 (Nr5a2) and Hnf4α (Nr2a1), which positively regulate the gene expression of Cyp7a1 and Cyp8b1. Moreover, all-trans RA induced the gene expression levels of negative regulators of bile acid synthesis including hepatic Fgfr4, Fxr, and Shp (Nr0b2) as well as ileal Fgf15. All-trans RA also decreased the expression of Abcb11 and Slc51b, which have a role in bile acid transport. Consistently, all-trans RA reduced hepatic bile acid levels and the ratio of CA/CDCA, as demonstrated by liquid chromatography-mass spectrometry. The data suggest that all-trans RA-induced SHP may contribute to the inhibition of CYP7A1 and CYP8B1, which in turn reduces bile acid synthesis and affects lipid absorption in the gastrointestinal tract. PMID:25175738

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.

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

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.

Trans -cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis –induced inflammation and virulence

PubMed Central

Rao, Vivek; Gao, Feng; Chen, Bing; Jacobs, William R.; Glickman, Michael S.

2006-01-01

Recent studies have shown that fine structural modifications of Mycobacterium tuberculosis cell envelope lipids mediate host cell immune activation during infection. One such alteration in lipid structure is cis-cyclopropane modification of the mycolic acids on trehalose dimycolate (TDM) mediated by proximal cyclopropane synthase of α mycolates (pcaA), a proinflammatory lipid modification during early infection. Here we examine the pathogenetic role and immunomodulatory function of mycolic acid cyclopropane stereochemistry by characterizing an M. tuberculosis cyclopropane–mycolic acid synthase 2 (cmaA2) null mutant (ΔcmaA2) that lacks trans-cyclopropanation of mycolic acids. Although titers of WT and ΔcmaA2 organisms were identical during mouse infection, ΔcmaA2 bacteria were hypervirulent while inducing larger granulomas than WT M. tuberculosis. The hypervirulence of the ΔcmaA2 strain depended on host TNF-α and IFN-γ. Loss of trans-cyclopropanation enhanced M. tuberculosis–induced macrophage inflammatory responses, a phenotype that was transferable with petroleum ether extractable lipids. Finally, purified TDM lacking trans-cyclopropane rings was 5-fold more potent in stimulating macrophages. These results establish cmaA2-dependent trans-cyclopropanation of TDM as a suppressor of M. tuberculosis–induced inflammation and virulence. In addition, cyclopropane stereochemistries on mycolic acids interact directly with host cells to both positively and negatively influence host innate immune activation. PMID:16741578

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

Trans-methylation reactions in plants: focus on the activated methyl cycle.

PubMed

Rahikainen, Moona; Alegre, Sara; Trotta, Andrea; Pascual, Jesús; Kangasjärvi, Saijaliisa

2018-02-01

Trans-methylation reactions are vital in basic metabolism, epigenetic regulation, RNA metabolism, and posttranslational control of protein function and therefore fundamental in determining the physiological processes in all living organisms. The plant kingdom is additionally characterized by the production of secondary metabolites that undergo specific hydroxylation, oxidation and methylation reactions to obtain a wide array of different chemical structures. Increasing research efforts have started to reveal the enzymatic pathways underlying the biosynthesis of complex metabolites in plants. Further engineering of these enzymatic machineries offers significant possibilities in the development of bio-based technologies, but necessitates deep understanding of their potential metabolic and regulatory interactions. Trans-methylation reactions are tightly coupled with the so-called activated methyl cycle (AMC), an essential metabolic circuit that maintains the trans-methylation capacity in all living cells. Tight regulation of the AMC is crucial in ensuring accurate trans-methylation reactions in different subcellular compartments, cell types, developmental stages and environmental conditions. This review addresses the organization and posttranslational regulation of the AMC and elaborates its critical role in determining metabolic regulation through modulation of methyl utilization in stress-exposed plants. © 2017 Scandinavian Plant Physiology Society.

Oxidized LDL activates blood platelets through CD36/NOX2–mediated inhibition of the cGMP/protein kinase G signaling cascade

PubMed Central

Magwenzi, Simbarashe; Woodward, Casey; Wraith, Katie S.; Aburima, Ahmed; Raslan, Zaher; Jones, Huw; McNeil, Catriona; Wheatcroft, Stephen; Yuldasheva, Nadira; Febbriao, Maria; Kearney, Mark

2015-01-01

Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36−/− murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2−/− mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3′,5′-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. PMID:25710879

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

Can we conserve trans-resveratrol content and antioxidant activity during industrial production of chocolate?

PubMed

Salvador, Izabela; Massarioli, Adna P; Silva, Anna Paula S; Malaguetta, Heloísa; Melo, Priscilla S; Alencar, Severino M

2018-05-23

Cocoa exhibits high content of phenolic compounds, among which trans-resveratrol stands out, associated with several bioactive activities such as antioxidant properties. Chocolate contains reduced amounts of these bioactive compounds due to losses during the production process. Therefore, this study aimed to assess changes in total phenolic content and specifically trans-resveratrol, as well as in the antioxidant activity of cocoa and its products during industrial production of chocolate. A total of ten different cocoa products were analyzed. The processes of fermentation and roasting caused significant loss of total phenolic compounds and antioxidant activity. However, due to high temperature, roasting had a major influence on this loss (71% for total phenolic compounds and 53% to 77% for antioxidant activity), except for trans-resveratrol. The content of trans-resveratrol formed after fermentation (9.8 μg kg -1 ) showed little variation during the processes, and it was detected in higher concentrations both in natural (11.4 μg kg -1 ) and in alkalized cocoa powder (13.5 μg kg -1 ). Alkalization of cocoa products led to loss of capacity of deactivating superoxide radical. These findings contribute to the optimization of the production process of chocolate and other food products containing cocoa and its derivatives aiming to better preserve their bioactive compounds. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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.

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

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

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.

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.

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

Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses.

PubMed Central

Everett, R D; Dunlop, M

1984-01-01

This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA isolated after infection. The results showed that Herpes simplex viruses 1 and 2, Pseudorabies virus, Variella Zoster virus, Human Cytomegalovirus, Equine herpes virus-1 and Adenovirus-2 activate transcription from both promoters tested. In contrast, SV40 did not activate transcription in trans in this assay. The possible mechanisms of this activation are discussed. Images PMID:6089105

[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.

Pin1-FADD interactions regulate Fas-mediated apoptosis in activated eosinophils#

PubMed Central

Oh, Jiyoung; Malter, James S.

2013-01-01

Abnormally long-lived eosinophils (Eos) are the major inflammatory component of allergic responses in the lungs of active asthmatics. Eos recruited to the airways after allergen exposure produce and respond to IL-5 and GM-CSF, enhancing their survival. Pro-survival signaling activates Pin1, a cis-trans peptidyl isomerase (PPIase) that binds to Bax and prevents it activation. How long-lived Eos, despite the continued presence of GM-CSF or IL-5, eventually undergo apoptosis to end allergic inflammation remains unclear. Here we show that Pin1 location, activity and protein interactions are jointly influenced by Fas and pro-survival cytokine IL-5. Fas signaling strongly induced the phosphorylation of FADD at Ser194 and Pin1 at Ser16 as well as their nuclear accumulation. Phospho-mimic Ser194Glu FADD mutants accelerated Eos apoptosis compared to WT or Ser194Ala mutants. Downstream of FADD phosphorylation, Caspase 8, 9 and 3 cleavage as well as Eos apoptosis induced by Fas were reduced by constitutively active Pin1 and enhanced by Pin1 inhibition. Pin1 was activated by IL-5 while simultaneous IL-5 and anti-Fas treatment modestly reduced PPIase activity but induced Pin1 to associate with FADD after its phosphorylation at Ser194. Mechanistically, Pin1 mediated isomerization facilitated the subsequent dephosphorylation of Ser194 FADD and maintenance of cytoplasmic location. In vivo activated bronchoalvelolar (BAL) Eos obtained after allergen challenge showed elevated survival and Pin1 activity that could be reversed by anti-Fas. Therefore, our data suggest that Pin1 is a critical link between FADD mediated cell death and IL-5 mediated pro-survival signaling. PMID:23606538

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

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

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.

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

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

PubMed

Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

2018-04-01

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

Synthesis of 2,3-trans-3,4-cis- and 2,3-trans-3,4-trans-2,3,4-triphenyltetrahydrofurans.

PubMed

Munshi, K L; Dikshit, D K; Kapil, R S; Anand, N

1974-04-01

The synthesis of 2,3-trans-3,4-cis- and 2,3-trans-3,4-trans-2,3,4-triphenyltetrahydrofurans was undertaken because these compounds incorportae the essential structural features of certain 2,3-diphenyl-benzofurans and 1,2,3-triphenylalkanones reported earlier to have marked antifertility activity. The synthesis of the 2 tetrahydrofurans was achieved by the cyclization of corresponding 2,3,4-triphenylbutane-1,4-diols upon heating with dimethyl sulfoxide (DMSO). The butane 1,4-diols were in turn prepared either by direct litium aluminum hydride (LAH) reduction of methyl 3-benzoyl-2,3-diphenylpropionates or by conversion of these propionates to delta-3,4-butryrolactones followed by LAH reduction. The propionates were prepared from the Fiedel-Crafts reaction of 2,3-diphenylsuccinic anhydride with benzene. Tetrahydrofurans were tested for their antiimplantation activity in rats. 2,3-trans-3,4-cis-2,4-diphenyl-3-p -(beta-pyrrolidinoethoxy) phenyltetrahydrofuran oxalate was found to inhibit implantation completely at 50 mg/kg, but was inefective at a lower dose.

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.

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.

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.

Antifungal activity of the essential oil of Illicium verum fruit and its main component trans-anethole.

PubMed

Huang, Yongfu; Zhao, Jianglin; Zhou, Ligang; Wang, Jihua; Gong, Youwen; Chen, Xujun; Guo, Zejian; Wang, Qi; Jiang, Weibo

2010-10-27

In order to identify natural products for plant disease control, the essential oil of star anise (Illicium verum Hook. f.) fruit was investigated for its antifungal activity on plant pathogenic fungi. The fruit essential oil obtained by hydro-distillation was analyzed for its chemical composition by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). trans-Anethole (89.5%), 2-(1-cyclopentenyl)-furan (0.9%) and cis-anethole (0.7%) were found to be the main components among 22 identified compounds, which accounted for 94.6% of the total oil. The antifungal activity of the oil and its main component trans-anethole against plant pathogenic fungi were determined. Both the essential oil and trans-anethole exhibited strong inhibitory effect against all test fungi indicating that most of the observed antifungal properties was due to the presence of trans-anethole in the oil, which could be developed as natural fungicides for plant disease control in fruit and vegetable preservation.

Nematicidal Activity of trans-2-Hexenal against Southern Root-Knot Nematode (Meloidogyne incognita) on Tomato Plants.

PubMed

Lu, Hongbao; Xu, Shuangyu; Zhang, Wenjuan; Xu, Chunmei; Li, Beixing; Zhang, Daxia; Mu, Wei; Liu, Feng

2017-01-25

Botanical nematicides have recently received increasing interest because of the high risks of some traditional nematicides to human health and the environment. This study evaluated the nematicidal activity of a plant volatile, trans-2-hexenal, against Meloidogyne incognita. This compound exhibited higher activity in a fumigation experiment than in the aqueous phase in vitro. Both in pot tests and in field trials, trans-2-hexenal showed significant efficacy against M. incognita while maintaining excellent plant growth, especially at doses of 1000 and 500 L ha -1 , which were superior to that of abamectin at 180 g ha -1 via hole application treatment but not significantly different from fumigation with 400 kg ha -1 of dazomet. Furthermore, plants treated with 500 L ha -1 trans-2-hexenal had fruit yields 20.2 and 45% greater than the control group. On this basis, trans-2-hexenal may be a potential alternative fumigation agent for controlling M. incognita on tomato crops.

EGFR mediates astragaloside IV-induced Nrf2 activation to protect cortical neurons against in vitro ischemia/reperfusion damages

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

Gu, Da-min; Lu, Pei-Hua, E-mail: lphty1_1@163.com; Zhang, Ke

In this study, we tested the potential role of astragaloside IV (AS-IV) against oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced damages in murine cortical neurons, and studied the associated signaling mechanisms. AS-IV exerted significant neuroprotective effects against OGD/R by reducing reactive oxygen species (ROS) accumulation, thereby attenuating oxidative stress and neuronal cell death. We found that AS-IV treatment in cortical neurons resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by Nrf2 Ser-40 phosphorylation, and its nuclear localization, as well as transcription of antioxidant-responsive element (ARE)-regulated genes: heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1) and sulphiredoxin 1 (SRXN-1). Knockdown of Nrf2 throughmore » lentiviral shRNAs prevented AS-IV-induced ARE genes transcription, and abolished its anti-oxidant and neuroprotective activities. Further, we discovered that AS-IV stimulated heparin-binding-epidermal growth factor (HB-EGF) release to trans-activate epidermal growth factor receptor (EGFR) in cortical neurons. Blockage or silencing EGFR prevented Nrf2 activation by AS-IV, thus inhibiting AS-IV-mediated anti-oxidant and neuroprotective activities against OGD/R. In summary, AS-IV protects cortical neurons against OGD/R damages through activating of EGFR-Nrf2 signaling. - Highlights: • Pre-treatment of astragaloside IV (AS-IV) protects murine cortical neurons from OGD/R. • AS-IV activates Nrf2-ARE signaling in murine cortical neurons. • Nrf2 is required for AS-IV-mediated anti-oxidant and neuroprotective activities. • AS-IV stimulates HB-EGF release to trans-activate EGFR in murine cortical neurons. • EGFR mediates AS-IV-induced Nrf2 activation and neuroprotection against OGD/R.« less

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.

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

Identification of trans-tiliroside as active principle with anti-hyperglycemic, anti-hyperlipidemic and antioxidant effects from Potentilla chinesis.

PubMed

Qiao, Wei; Zhao, Chuan; Qin, Nan; Zhai, Hui Yuan; Duan, Hong Quan

2011-05-17

The present study was carried out to isolate and identify trans-tiliroside as principal compound with anti-hyperglycemic, anti-hyperlipidemic and antioxidant effects from Potentilla chinesis. A bioactive compound, trans-tiliroside was isolated from the ethanol extract of Potentilla chinesis and its administration dose was optimized and patented. The normal, alloxan-induced diabetic mice and streptozotocin-induced diabetic rats were used to evaluate the anti-hyperglycemic, anti-hyperlipidemic and antioxidant effects of trans-tiliroside from Potentilla chinesis. Biochemical parameters were assayed in blood samples of different groups of alloxan-induced diabetic mice and streptozotocin-induced diabetic rats. The level of fasting serum glucose levels, triglycerides (TG) and total cholesterol (TC) in alloxan-induced diabetic mice were significantly decrease after daily oral administration of trans-tiliroside in normal and diabetic mice at doses of 0.4, 0.8 and 1.6 mg/kg/day, for 15 days. Blood glucose level was significant decrease in STZ induced diabetic rats by trans-tiliroside (1.2 and 0.3mg/kg body weight for 10 weeks). The content of TC, low density lipoprotein (LDL-C) and TG levels were decreased and high density lipoprotein (HDL-C) content was increased, so lipid metabolism was improved. Moreover, trans-tiliroside revealed antioxidant activity as shown by increased activities of superoxide dismutase (SOD), decreased level of malondialdehyde (MDA) in diabetic rats. Histological morphology examination showed that the trans-tiliroside restored the damage of pancreas tissues in rats with diabetes mellitus. Trans-tiliroside, a constituent from Potentilla chinesis, revealed significant anti-hyperglycemic, anti-hyperlipidemic and antioxidant activities. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

PubMed

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

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

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

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.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

PubMed Central

Wong, J G; Smithgall, M D; Haffar, O K

1997-01-01

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL. Images Figure 1 Figure 2 Figure 3 PMID:9135558

GPER Mediates Functional Endothelial Aging in Renal Arteries.

PubMed

Meyer, Matthias R; Rosemann, Thomas; Barton, Matthias; Prossnitz, Eric R

2017-01-01

Aging is associated with impaired renal artery function, which is partly characterized by arterial stiffening and a reduced vasodilatory capacity due to excessive generation of reactive oxygen species by NADPH oxidases (Nox). The abundance and activity of Nox depends on basal activity of the heptahelical transmembrane receptor GPER; however, whether GPER contributes to age-dependent functional changes in renal arteries is unknown. This study investigated the effect of aging and Nox activity on renal artery tone in wild-type and GPER-deficient (Gper-/-) mice (4 and 24 months old). In wild-type mice, aging markedly impaired endothelium-dependent, nitric oxide (NO)-mediated relaxations to acetylcholine, which were largely preserved in renal arteries of aged Gper-/- mice. The Nox inhibitor gp91ds-tat abolished this difference by greatly enhancing relaxations in wild-type mice, while having no effect in Gper-/- mice. Contractions to angiotensin II and phenylephrine in wild-type mice were partly sensitive to gp91ds-tat but unaffected by aging. Again, deletion of GPER abolished effects of Nox inhibition on contractile responses. In conclusion, basal activity of GPER is required for the age-dependent impairment of endothelium-dependent, NO-mediated relaxation in the renal artery. Restoration of relaxation by a Nox inhibitor in aged wild-type but not Gper-/- mice strongly supports a role for Nox-derived reactive oxygen species as the underlying cause. Pharmacological blockers of GPER signaling may thus be suitable to inhibit functional endothelial aging of renal arteries by reducing Nox-derived oxidative stress and, possibly, the associated age-dependent deterioration of kidney function. © 2017 S. Karger AG, Basel.

Human Mucosal Mast Cells Capture HIV-1 and Mediate Viral trans-Infection of CD4+ T Cells.

PubMed

Jiang, Ai-Ping; Jiang, Jin-Feng; Wei, Ji-Fu; Guo, Ming-Gao; Qin, Yan; Guo, Qian-Qian; Ma, Li; Liu, Bao-Chi; Wang, Xiaolei; Veazey, Ronald S; Ding, Yong-Bing; Wang, Jian-Hua

2015-12-30

The gastrointestinal mucosa is the primary site where human immunodeficiency virus type 1 (HIV-1) invades, amplifies, and becomes persistently established, and cell-to-cell transmission of HIV-1 plays a pivotal role in mucosal viral dissemination. Mast cells are widely distributed in the gastrointestinal tract and are early targets for invasive pathogens, and they have been shown to have increased density in the genital mucosa in HIV-infected women. Intestinal mast cells express numerous pathogen-associated molecular patterns (PAMPs) and have been shown to combat various viral, parasitic, and bacterial infections. However, the role of mast cells in HIV-1 infection is poorly defined. In this study, we investigated their potential contributions to HIV-1 transmission. Mast cells isolated from gut mucosal tissues were found to express a variety of HIV-1 attachment factors (HAFs), such as DC-SIGN, heparan sulfate proteoglycan (HSPG), and α4β7 integrin, which mediate capture of HIV-1 on the cell surface. Intriguingly, following coculture with CD4(+) T cells, mast cell surface-bound viruses were efficiently transferred to target T cells. Prior blocking with anti-HAF antibody or mannan before coculture impaired viral trans-infection. Cell-cell conjunctions formed between mast cells and T cells, to which viral particles were recruited, and these were required for efficient cell-to-cell HIV-1 transmission. Our results reveal a potential function of gut mucosal mast cells in HIV-1 dissemination in tissues. Strategies aimed at preventing viral capture and transfer mediated by mast cells could be beneficial in combating primary HIV-1 infection. In this study, we demonstrate the role of human mast cells isolated from mucosal tissues in mediating HIV-1 trans-infection of CD4(+) T cells. This finding facilitates our understanding of HIV-1 mucosal infection and will benefit the development of strategies to combat primary HIV-1 dissemination. Copyright © 2016, American Society

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.

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.

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

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.

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.

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

Synthesis and antioxidant activity of polyhydroxylated trans-restricted 2-arylcinnamic acids.

PubMed

Miliovsky, Mitko; Svinyarov, Ivan; Prokopova, Elena; Batovska, Daniela; Stoyanov, Simeon; Bogdanov, Milen G

2015-02-02

A series of sixteen polyhydroxylated trans-restricted 2-arylcinnamic acid analogues 3a-p were synthesized through a one-pot reaction between homophthalic anhydrides and various aromatic aldehydes, followed by treatment with BBr3. The structure of the newly synthesized compounds was confirmed by spectroscopic methods and the configuration around the double bond was unequivocally estimated by means of gated decoupling 13C-NMR spectra. It was shown that the trans-cinnamic acid fragment incorporated into the target compounds' structure ensures the cis-configuration of the stilbene backbone and prevents further isomerization along the carbon-carbon double bond. The antioxidant activity of compounds 3a-p was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH●), hydroxyl (OH●) and superoxide (O2●▬) radicals. The results obtained showed that the tested compounds possess higher activities than natural antioxidants such as protocatechuic acid, caffeic acid and gallic acid. Moreover, it was shown that a combination of two different and independently acting fragments of well-known pharmacological profiles into one covalently bonded hybrid molecule evoke a synergistic effect resulting in higher than expected activity. To rationalize the apparent antioxidant activity and to establish the mechanism of action, a SAR analysis and DFT quantum chemical computations were also performed.

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

Application of antibody-mediated extraction for the stereoselective determination of the active metabolite of loxoprofen in human and rat plasma.

PubMed

Takasaki, W; Tanaka, Y

1992-01-01

Antibody-mediated extraction followed by chiral high-performance liquid chromatography (HPLC) was applied to stereoselective determination in human and rat plasma of the active metabolite [(2S,1'R,2'S)-trans-alcohol] with three chiral centers of Loxoprofen, a 2-arylpropionic acid antiinflammatory agent after oral administration. Antiserum against the (1'R,2'S)-cyclopentanol moiety was obtained from a rabbit immunized with bovine serum albumin conjugate linked to the propionic acid moiety, in which another chiral center is located. Then, the immunoglobulin G purified by a protein A column was coupled to BrCN-activated Sepharose 4B. Plasma samples were applied to the immobilized antibody column. After washing the column to remove unrequired stereoisomers, a mixture of two diastereomers whose configurations were 1'R,2'S in the cyclopentanol moiety was extracted with 95% methanol. The solvent was evaporated and the residue was derivatized with (+)-(R)-1-(1-naphthyl)ethylamine as a chiral reagent to separate the diastereomers by HPLC. This combined analytical method showed the stereoselective metabolism of Loxoprofen in human, that is, 64% of the total amount of four trans-alcohol stereoisomers was in the 2S,1'R,2'S form, which is the active metabolite. This phenomenon was also observed in rats given Loxoprofen and its (2S)- and (2R)-isomers, and is explained by stereoselective ketone reduction of Loxoprofen to the (1'R,2'S)-trans-alcohol and inversion from 2R to 2S in the propionic acid moiety. Antibody-mediated extraction should be a selective and simple clean-up method for determining haptens with complicated structures, combined with an appropriate analytical method.

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.

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

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.…

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

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

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.

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

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

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

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

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.

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.

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.

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.

Trans-membrane Signaling in Photosynthetic State Transitions

PubMed Central

Singh, Sandeep K.; Hasan, S. Saif; Zakharov, Stanislav D.; Naurin, Sejuti; Cohn, Whitaker; Ma, Jia; Whitelegge, Julian P.; Cramer, William A.

2016-01-01

Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution (“state transition”) of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation. PMID:27539852

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

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.

Trans3D: a free tool for dynamical visualization of EEG activity transmission in the brain.

PubMed

Blinowski, Grzegorz; Kamiński, Maciej; Wawer, Dariusz

2014-08-01

The problem of functional connectivity in the brain is in the focus of attention nowadays, since it is crucial for understanding information processing in the brain. A large repertoire of measures of connectivity have been devised, some of them being capable of estimating time-varying directed connectivity. Hence, there is a need for a dedicated software tool for visualizing the propagation of electrical activity in the brain. To this aim, the Trans3D application was developed. It is an open access tool based on widely available libraries and supporting both Windows XP/Vista/7(™), Linux and Mac environments. Trans3D can create animations of activity propagation between electrodes/sensors, which can be placed by the user on the scalp/cortex of a 3D model of the head. Various interactive graphic functions for manipulating and visualizing components of the 3D model and input data are available. An application of the Trans3D tool has helped to elucidate the dynamics of the phenomena of information processing in motor and cognitive tasks, which otherwise would have been very difficult to observe. Trans3D is available at: http://www.eeg.pl/. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid.

PubMed

Walczak, H A; Dean, J V

2000-02-01

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.

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

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

PubMed

Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

2015-07-15

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

PubMed

Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

2009-02-24

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

Approaching an experimental electron density model of the biologically active trans -epoxysuccinyl amide group-Substituent effects vs. crystal packing

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

Shi, Ming W.; Stewart, Scott G.; Sobolev, Alexandre N.

The trans-epoxysuccinyl amide group as a biologically active moiety in cysteine protease inhibitors such as loxistatin acid E64c has been used as a benchmark system for theoretical studies of environmental effects on the electron density of small active ingredients in relation to their biological activity. Here, the synthesis and the electronic properties of the smallest possible active site model compound are reported to close the gap between the unknown experimental electron density of trans-epoxysuccinyl amides and the well-known function of related drugs. Intramolecular substituent effects are separated from intermolecular crystal packing effects on the electron density, which allows us tomore » predict the conditions under which an experimental electron density investigation on trans-epoxysuccinyl amides will be possible. In this context, the special importance of the carboxylic acid function in the model compound for both crystal packing and biological activity is revealed through the novel tool of model energy analysis.« less

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.

Homologous SV40 RNA trans-splicing

PubMed Central

Eul, Joachim; Patzel, Volker

2013-01-01

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5′ donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5′ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5′ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes. PMID:24178438

Understanding How Sexual and Gender Minority Stigmas Influence Depression Among Trans Women and Men Who Have Sex with Men in India.

PubMed

Chakrapani, Venkatesan; Vijin, Pandara Purayil; Logie, Carmen H; Newman, Peter A; Shunmugam, Murali; Sivasubramanian, Murugesan; Samuel, Miriam

2017-06-01

Few studies have assessed how sexual and gender minority stigmas affect the mental health of trans women and self-identified men who have sex with men (MSM) in India, populations with a high HIV burden. We tested whether social support and resilient coping act as mediators of the effect of sexual and gender minority stigmas on depression as proposed by Hatzenbuehler's psychological mediation framework, or as moderators based on Meyer's minority stress theory. We conducted a cross-sectional survey among trans women (n = 300) and MSM (n = 300) recruited from urban and rural sites in India. Standardized scales were used to measure depression (outcome variable), transgender identity stigma/MSM stigma (predictor variables), and social support and resilient coping (tested as moderators and parallel mediators). The mediation and moderation models were tested separately for trans women and MSM, using Hayes' PROCESS macro in SPSS. Participants' mean age was 29.7 years (standard deviation 8.1). Transgender identity stigma and MSM stigma were significant predictors (significant total and direct effects) of depression, as were social support and resilient coping. Among trans women and MSM, social support and resilient coping mediated (i.e., significant specific indirect effects), but did not moderate, the effect of stigma on depression, supporting the psychological mediation framework. Sexual and gender minority stigmas are associated with depression, with social support and resilient coping as mediators. In addition to stigma reduction interventions at the societal level, future interventions should focus on improving social support and promoting resilience among trans women and MSM in India.

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

Benzoic Acid Derivatives with Trypanocidal Activity: Enzymatic Analysis and Molecular Docking Studies toward Trans-Sialidase.

PubMed

Kashif, Muhammad; Moreno-Herrera, Antonio; Villalobos-Rocha, Juan Carlos; Nogueda-Torres, Benjamín; Pérez-Villanueva, Jaime; Rodríguez-Villar, Karen; Medina-Franco, José Lius; de Andrade, Peterson; Carvalho, Ivone; Rivera, Gildardo

2017-10-30

Chagas, or American trypanosomiasis, remains an important public health problem in developing countries. In the last decade, trans -sialidase has become a pharmacological target for new anti-Chagas drugs. In this work, the aims were to design and find a new series of benzoic acid derivatives as trans -sialidase (TS) inhibitors and anti-trypanosomal agents. Three compounds ( 14 , 18 , and 19 ) sharing a para -aminobenzoic acid moiety showed more potent trypanocidal activity than the commercially available drugs nifurtimox and benznidazole in both strains: the lysis concentration of 50% of the population (LC 50 ) was <0.15 µM on the NINOA strain, and LC 50 < 0.22 µM on the INC-5 strain. Additionally, compound 18 showed a moderate inhibition (47%) on the trans -sialidase enzyme and a binding model similar to DANA (pattern A).

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

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

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

PubMed Central

Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

2016-01-01

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

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.

Boycunts and bonus holes: trans men's bodies, neoliberalism, and the sexual productivity of genitals.

PubMed

Edelman, Elijah Adiv; Zimman, Lal

2014-01-01

Recent theorizations of trans embodiment have brought attention to the ways neoliberalism limits the productivity of nonnormatively gendered bodies. This article deals with the discursive framing of embodiment and sexual desirability among trans men and other transmasculine persons negotiating Internet-mediated homoerotic spaces. Micro-level analysis of discourse structure and macro-level analysis of socio-political context together show how trans men navigate homonormative sexual economies by linguistically recuperating their bodies' sexually productivity. Instead of undermining claims of embodied masculinity and homoerotic value, potential sites of exclusion-i.e., trans genitals-become sites of flexible accumulation that enhance rather than detract from their bearers' desirability.

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

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

Dehalogenation Activity of Selected Fungi Toward δ-Iodo-γ-Lactone Derived from trans,trans-Farnesol.

PubMed

Gliszczyńska, Anna; Gładkowski, Witold; Świtalska, Marta; Wietrzyk, Joanna; Szumny, Antoni; Gębarowska, Elżbieta; Wawrzeńczyk, Czesław

2016-04-01

Time-course of biotransformation of racemic trans-4-((E)-4',8'-dimethylnona-3',7'-dien-1-yl)-5-iodomethyl-4-methyldihydrofuran-2-one (1) in fungal and yeast cultures was investigated. In these conditions, the substrate 1 was enantioselectively dehalogenated yielding 4-((E)-4',8'-dimethylnona-3',7'-dien-1-yl)-4-methyl-5-methylenedihydrofuran-2-one (2) and its structure was established based on the spectroscopic data. The most effective biocatalyst used was Didymosphaeria igniaria, which catalyzed the process with highest rate and enantioselectivity (ee of product = 76%). The antiproliferative activity of δ-iodo-γ-lactone 1, product of its biotransformation 2, and starting substrate (farnesol) were evaluated toward two cancer cell lines: A549 (human lung adenocarcinoma) and HL-60 (human promyelocytic leukemia). © 2016 Verlag Helvetica Chimica Acta AG, Zürich.

Tryptophan as a molecular shovel in the glycosyl transfer activity of Trypanosoma cruzi trans-sialidase.

PubMed

Mitchell, Felicity L; Miles, Steven M; Neres, João; Bichenkova, Elena V; Bryce, Richard A

2010-05-19

Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp(312) loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp(312) flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Local sources of retinoic acid coincide with retinoid-mediated transgene activity during embryonic development.

PubMed Central

Colbert, M C; Linney, E; LaMantia, A S

1993-01-01

We have assessed whether retinoic acid (RA) comes from local sources or is available widely to activate gene expression in embryos. We used an RA-responsive indicator cell line, L-C2A5, to localize RA sources. In these cells, an RA-sensitive promoter/lacZ reporter construct used previously by us to produce indicator transgenic mice is induced globally by RA in medium or locally by RA released at physiological concentrations (1 nM) from AG-1X2 resin beads. Furthermore, the cells are differentially responsive to the 9-cis and all-trans isomers of RA at low concentrations. Indicator transgenic mice with the same promoter/reporter construct were used to identify regions of RA-mediated gene activation. There are distinct domains of lacZ expression in the cervical and lumbar spinal cords of embryonic indicator mice. This pattern might reflect localized RA sources or restricted spatial and temporal expression of RA receptors, binding proteins, or other factors. To resolve this issue we compared the pattern of transgene activation in indicator cell monolayers cocultured with normal embryonic spinal cords with that in transgenic spinal cords. The explants induced reporter gene expression in L-C2A5 monolayers in a pattern identical to that in transgenic mice: alar regions of the cervical and lumbar cord were positive whereas those in the thoracic and sacral regions were not. We conclude that restricted sources of RA in the developing spinal cord mediate the local activation of RA-inducible genes. Thus, region-specific gene activation in embryos can be mediated by precisely localized sources of inductive molecules like RA. Images Fig. 1 Fig. 2 Fig. 3 PMID:8341670

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

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

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.

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…

Tryptophan as a Molecular Shovel in the Glycosyl Transfer Activity of Trypanosoma cruzi Trans-sialidase

PubMed Central

Mitchell, Felicity L.; Miles, Steven M.; Neres, João; Bichenkova, Elena V.; Bryce, Richard A.

2010-01-01

Abstract Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp312 loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp312 flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease. PMID:20441732

The antidepressant-like effect of trans-astaxanthin involves the serotonergic system.

PubMed

Jiang, Xi; Zhu, Keqi; Xu, Quanyi; Wang, Guokang; Zhang, Jiajia; Cao, Rongrong; Ye, Jiang; Yu, Xuefeng

2017-04-11

The antidepressant-like effect of trans-astaxanthin, a compound present rich in algae, was evaluated through behavioral and neurochemical methods. Results showed that trans-astaxanthin treatment significantly decreased the immobility time in force swim test and tail suspension test, but did not influence locomotor activity. Trans-astaxanthin treatment did not effectively antagonize hypothermia and ptosis induced by reserpine. However, pre-treatment with para-chlorophenylalanine abolished the anti-immobility effect of trans-astaxanthin in force swim and tail suspension test. These results suggested that the mechanism of antidepressant-like effect of trans-astaxanthin may involve the serotonergic system, but not noradrenaline system. This hypothesis was confirmed by neurochemical assays which showed that trans-astaxanthin increased serotonin levels in the hippocampus, frontal cortex, striatum and hypothalamus. Furthermore, our data suggested that trans-astaxanthin decreased indoleamine 2, 3-dioxygenase activity in the hippocampus, frontal cortex and hypothalamus. Inhibition of indoleamine 2,3-dioxygenase activity subsequently decreased the kynurenine/tryptophan ratio and increased the serotonin/tryptophan ratio in these brain regions. Taken together, these findings indicate that the antidepressant-like effect of trans-astaxanthin involves the serotonergic system.

The antidepressant-like effect of trans-astaxanthin involves the serotonergic system

PubMed Central

Jiang, Xi; Zhu, Keqi; Xu, Quanyi; Wang, Guokang; Zhang, Jiajia; Cao, Rongrong; Ye, Jiang; Yu, Xuefeng

2017-01-01

The antidepressant-like effect of trans-astaxanthin, a compound present rich in algae, was evaluated through behavioral and neurochemical methods. Results showed that trans-astaxanthin treatment significantly decreased the immobility time in force swim test and tail suspension test, but did not influence locomotor activity. Trans-astaxanthin treatment did not effectively antagonize hypothermia and ptosis induced by reserpine. However, pre-treatment with para-chlorophenylalanine abolished the anti-immobility effect of trans-astaxanthin in force swim and tail suspension test. These results suggested that the mechanism of antidepressant-like effect of trans-astaxanthin may involve the serotonergic system, but not noradrenaline system. This hypothesis was confirmed by neurochemical assays which showed that trans-astaxanthin increased serotonin levels in the hippocampus, frontal cortex, striatum and hypothalamus. Furthermore, our data suggested that trans-astaxanthin decreased indoleamine 2, 3-dioxygenase activity in the hippocampus, frontal cortex and hypothalamus. Inhibition of indoleamine 2,3-dioxygenase activity subsequently decreased the kynurenine/tryptophan ratio and increased the serotonin/tryptophan ratio in these brain regions. Taken together, these findings indicate that the antidepressant-like effect of trans-astaxanthin involves the serotonergic system. PMID:28424423

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.

Novel trans-Ferulic Acid Derivatives Containing a Chalcone Moiety as Potential Activator for Plant Resistance Induction.

PubMed

Gan, Xiuhai; Hu, Deyu; Wang, Yanjiao; Yu, Lu; Song, Baoan

2017-06-07

A series of novel trans-ferulic acid derivatives containing a chalcone moiety were designed and synthesized to induce plant resistance. Antiviral activities of the compounds were evaluated. Bioassay results demonstrated that compounds F3, F6, F17, and F27 showed remarkable curative, protective, and inactivating activities against tobacco mosaic virus (TMV). With a 50% effective concentration (EC 50 ) value of 98.78 μg mL -1 , compound F27 exhibited the best protective activity compared with trans-ferulic acid (328.6 μg mL -1 ), dufulin (385.6 μg mL -1 ), and ningnanmycin (241.3 μg mL -1 ). This protective ability was associated with potentiation of defense-related enzyme activity and activation of photosynthesis of tobacco at an early stage. This notion was confirmed by up-regulated expression of stress responses and photosynthesis regulating proteins. This work revealed that F27 can induce resistance and enhance plant tolerance to TMV infection. Hence, F27 can be considered as a novel activator for inducing plant resistance.

Trans-synaptic zinc mobilization improves social interaction in two mouse models of autism through NMDAR activation.

PubMed

Lee, Eun-Jae; Lee, Hyejin; Huang, Tzyy-Nan; Chung, Changuk; Shin, Wangyong; Kim, Kyungdeok; Koh, Jae-Young; Hsueh, Yi-Ping; Kim, Eunjoon

2015-05-18

Genetic aspects of autism spectrum disorders (ASDs) have recently been extensively explored, but environmental influences that affect ASDs have received considerably less attention. Zinc (Zn) is a nutritional factor implicated in ASDs, but evidence for a strong association and linking mechanism is largely lacking. Here we report that trans-synaptic Zn mobilization rapidly rescues social interaction in two independent mouse models of ASD. In mice lacking Shank2, an excitatory postsynaptic scaffolding protein, postsynaptic Zn elevation induced by clioquinol (a Zn chelator and ionophore) improves social interaction. Postsynaptic Zn is mainly derived from presynaptic pools and activates NMDA receptors (NMDARs) through postsynaptic activation of the tyrosine kinase Src. Clioquinol also improves social interaction in mice haploinsufficient for the transcription factor Tbr1, which accompanies NMDAR activation in the amygdala. These results suggest that trans-synaptic Zn mobilization induced by clioquinol rescues social deficits in mouse models of ASD through postsynaptic Src and NMDAR activation.

CD22ΔE12 as a molecular target for corrective repair using a RNA trans-splicing strategy: Anti-leukemic activity of a rationally designed RNA trans-splicing molecule

PubMed Central

Uckun, Fatih M.; Qazi, Sanjive; Ma, Hong; Reaman, Gregory H.; Mitchell, Lloyd G.

2015-01-01

Our recent studies have demonstrated that the CD22 exon 12 deletion (CD22ΔE12) is a characteristic genetic defect of therapy-refractory clones in pediatric B-precursor acute lymphoblastic leukemia (BPL) and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL. The purpose of the present study was to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for corrective repair using RNA trans-splicing therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide experimental evidence that the correction of the CD22ΔE12 genetic defect in human CD22ΔE12+ BPL cells using a rationally designed CD22 RNA trans-splicing molecule (RTM) caused a pronounced reduction of their clonogenicity. The RTM-mediated correction replaced the downstream mutation-rich segment of Intron 12 and remaining segments of the mutant CD22 pre-mRNA with wildtype CD22 Exons 10-14, thereby preventing the generation of the cis-spliced aberrant CD22ΔE12 product. The anti-leukemic activity of this RTM against BPL xenograft clones derived from CD22ΔE12+ leukemia patients provides the preclinical proof-of-concept that correcting the CD22ΔE12 defect with rationally designed CD22 RTMs may provide the foundation for therapeutic innovations that are needed for successful treatment of high-risk and relapsed BPL patients. PMID:25567759

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

PubMed Central

Liu, Xin; Ropp, Susan L.; Jackson, Richard J.; Frey, Teryl K.

1998-01-01

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn2+, Cd2+, and Co2+, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans. PMID:9557742

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest.

PubMed

Bortolotto, Luis Felipe Buso; Barbosa, Flávia Regina; Silva, Gabriel; Bitencourt, Tamires Aparecida; Beleboni, Rene Oliveira; Baek, Seung Joon; Marins, Mozart; Fachin, Ana Lúcia

2017-01-01

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA 1 ) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis. Copyright © 2016. Published by Elsevier Masson SAS.

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.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.

PubMed

Helledie, T; Antonius, M; Sorensen, R V; Hertzel, A V; Bernlohr, D A; Kølvraa, S; Kristiansen, K; Mandrup, S

2000-11-01

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.

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

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.

Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

PubMed

Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

2010-03-01

trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

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

The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter.

PubMed

Hoffmeister, Albrecht; Ropolo, Alejandro; Vasseur, Sophie; Mallo, Gustavo V; Bodeker, Hans; Ritz-Laser, Beate; Dressler, Gregory R; Vaccaro, Maria Ines; Dagorn, Jean-Charles; Moreno, Silvia; Iovanna, Juan Lucio

2002-06-21

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

Chromatin signaling to kinetochores: Trans-regulation of Dam1 methylation by histone H2B ubiquitination

PubMed Central

Latham, John A.; Chosed, Renée J.; Wang, Shanzhi; Dent, Sharon Y.R.

2011-01-01

Summary Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory cross-talk in trans between a histone modification and modification on a non-histone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries. PMID:21884933

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.

Synthesis of 7-azabicyclo[2.2.1]heptane and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives by base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides.

PubMed

Gómez-Sanchez, Elena; Soriano, Elena; Marco-Contelles, José

2007-11-09

We have studied the base-promoted heterocyclization of alkyl N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)carbamates and N-(cis(trans)-3,trans(cis)-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamides, investigating the effect of the nitrogen protecting group and the relative configuration of the leaving group at C3 and C4 on the outcome of this reaction. We have observed that the sodium hydride-promoted heterocyclization of alkyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamates (10, 12, 14, 16, 18) is a convenient method for the synthesis of 7-azabicyclo[2.2.1]heptane derivatives. For instance, the reaction of tert-butyl N-(cis-3,trans-4-dibromocyclohex-1-yl)carbamate (10) with sodium hydride in DMF at room temperature provides 2-bromo-7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]heptane (2) (52% yield), whose t-BuOK-promoted hydrogen bromide elimination affords 7-[(tert-butoxy)carbonyl]-7-azabicyclo[2.2.1]hept-2-ene (31) in 78% yield, an intermediate in the total synthesis of epibatidine (1). However, the NaH/DMF-mediated heterocyclization of alkyl N-(trans-3,cis-4-dibromocyclohex-1-yl)carbamates (11, 13) is a more structure dependent reaction, where the nucleophilic attack of the oxygen atom of the protecting group controls the outcome of the reaction, giving rise to benzooxazolone and 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, from low to moderate yields, in complex reaction mixtures. Conversely, the NaH/DMF heterocyclizations of N-(cis-3,trans-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (40) or N-(trans-3,cis-4-dibromocyclohex-1-yl)-2,2,2-trifluoroacetamide (42) are very clean reactions giving 7-azabicyclo[2.2.1]heptane or 2-oxa-4-azabicyclo[3.3.1]non-3-ene derivatives, respectively, in good yields. Finally, a mechanistic investigation, based on DFT calculations, has been carried out to rationalize the formation of the different adducts.

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.

Analysis of Photosynthetic Antenna Function in a Mutant of Arabidopsis thaliana (L.) Lacking trans-Hexadecenoic Acid 1

PubMed Central

McCourt, Peter; Browse, John; Watson, Jan; Arntzen, Charles J.; Somerville, Chris R.

1985-01-01

Several lines of evidence support the proposal that the unusual chloroplast-specific lipid acyl group Δ3,trans-hexadecenoic acid (trans-C16:1) stimulates the formation or maintenance of the oligomeric form of the light-harvesting chlorophyll a/b complex (LHCP). To assess the functional significance of this apparent association we have analyzed LHCP structure and function in a mutant of Arabidopsis thaliana (L.) which lacks trans-C16:1 by electrophoretic analysis of the protein-chlorophyll complexes and by measurements of chlorophyll fluorescence under a variety of conditions. By these criteria the putative oligomeric form of LHCP appears to be slightly more labile to detergent-mediated dissociation in the mutant. The oligomeric PSI chlorophyll-protein complex, associated with PSI, was also more labile to detergent-mediated dissociation in the mutant, suggesting a previously unsuspected association of trans-C16:1 with the PSI complex. However, no significant effect of the mutation on the efficiency of energy transfer from LHCP to the photochemical reaction centers was observed under any of the various conditions imposed. Also, the stability of the chlorophyll-protein complexes to temperature-induced dissociation was unaffected in the mutant. The role of trans-C16:1 is very subtle or is only conditionally expressed. Images Fig. 1 PMID:16664340

The trans-activator RNF12 and cis-acting elements effectuate X chromosome inactivation independent of X-pairing.

PubMed

Barakat, Tahsin Stefan; Loos, Friedemann; van Staveren, Selma; Myronova, Elvira; Ghazvini, Mehrnaz; Grootegoed, J Anton; Gribnau, Joost

2014-03-20

X chromosome inactivation (XCI) in female placental mammals is a vital mechanism for dosage compensation between X-linked and autosomal genes. XCI starts with activation of Xist and silencing of the negative regulator Tsix, followed by cis spreading of Xist RNA over the future inactive X chromosome (Xi). Here, we show that XCI does not require physical contact between the two X chromosomes (X-pairing) but is regulated by trans-acting diffusible factors. We found that the X-encoded trans-acting and dose-dependent XCI-activator RNF12 acts in concert with the cis-regulatory region containing Jpx, Ftx, and Xpr to activate Xist and to overcome repression by Tsix. RNF12 acts at two subsequent steps; two active copies of Rnf12 drive initiation of XCI, and one copy needs to remain active to maintain XCI toward establishment of the Xi. This two-step mechanism ensures that XCI is very robust and fine-tuned, preventing XCI of both X chromosomes. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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.

Distinct Conformations of Ly49 Natural Killer Cell Receptors Mediate MHC Class I Recognition in Trans and Cis

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

Back, J.; Malchiodi, E; Cho, S

2009-01-01

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors andmore » explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.« less

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.

Proceedings from the NIMH symposium on "NeuroAIDS in Africa: neurological and neuropsychiatric complications of HIV".

PubMed

Buch, Shilpa; Chivero, Ernest T; Hoare, Jackie; Jumare, Jibreel; Nakasujja, Noeline; Mudenda, Victor; Paul, Robert; Kanmogne, Georgette D; Sacktor, Ned; Wood, Charles; Royal, Walter; Joseph, Jeymohan

2016-10-01

Despite major advances in HIV-1 treatment, the prevalence of HIV-associated neurocognitive disorders (HAND) remains a problem, particularly as individuals on suppressive treatment continue to live longer. To facilitate discussion on emerging and future directions in HAND research, a meeting was held in Durban, South Africa in March 2015 as part of the Society of Neuroscientists of Africa (SONA) conference. The objective of the meeting was to assess the impact of HIV subtype diversity on HAND and immunological dysfunction. The meeting brought together international leaders in the area of neurological complications of HIV-1 infection with special focus on the African population. Research presentations indicated that HAND was highly prevalent and that inflammatory cytokines and immune-activation played important roles in progression of neurocognitive impairment. Furthermore, children on antiretroviral therapy were also at risk for developing neurocognitive impairment. With respect to the effect of HIV-1 subtype diversity, analyses of HIV-1 clade C infection among South Africans revealed that clade C infection induced cognitive impairment that was independent of the substitution in HIV-1 Trans-Activator of Transcription (Tat; C31S). At the cellular level, a Zambian study showed that clade C infection resulted in reduced brain cell death compared with clade B infection suggesting clade specific variations in mediating brain cell injury. Furthermore, ex vivo Tat protein from clade CRF02_AG, prevalent in West/ Central Africa, exhibited reduced disruption of brain endothelium compared with clade B Tat protein. Discussions shed light on future research directions aimed at understanding biomarkers and disease mechanisms critical for HAND.

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.

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

Comparing Trans-Spectrum and Same-Sex-Attracted Youth in Australia: Increased Risks, Increased Activisms

ERIC Educational Resources Information Center

Jones, Tiffany; Hillier, Lynne

2013-01-01

Tran-spectrum youth include those who are gender questioning, transgender, intersex, genderqueer, and androgynous. Drawing on data from an Australian study of more than 3,000 same-sex-attracted and trans-spectrum youth aged 14 to 21, this article compares a group of 91 trans-spectrum youth from the study to "cisgender" same-sex-attracted…

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

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

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

[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

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.

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

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

Wang, S.-F.; Huang, Jason C.; AIDS Prevention and Research Center, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan

2008-09-05

DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing {alpha}-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capablemore » of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.« less

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

Trans fatty acid isomers and the trans-9/trans-11 index in fat containing foods

PubMed Central

Kuhnt, Katrin; Baehr, Melanie; Rohrer, Carsten; Jahreis, Gerhard

2011-01-01

To determine trans fatty acid (TFA) distribution of contemporary foods, especially regarding individual trans octadecenoic acids (trans C18:1), 339 German foods of six categories (semi-solid fats, deep-fried potato products, bakery products, confectioneries, instant products and butter) were analysed using two GC methods. Results showed a high variation of TFA content between and within the categories containing between 0 and 40.5% of FAME except in butter, which is a source of natural TFA. The mean TFA values were below 2.0% of FAME, however, bakery products contained 4.5% and butter fat 3.2%, respectively. In addition, the distribution of individual trans C18:1 differed. In samples containing ruminant fat (butter and various confectioneries), vaccenic acid (t11-C18:1, t11) predominated, while in foods containing industrially hydrogenated fats, elaidic acid (trans-9, t9-) and t10-C18:1 were the major trans isomers.. This was reflected by a low t9/t11 index of 0.3 and 0.5 in butter and ruminant fat containing confectioneries, respectively, whilst the highest index was observed in shortenings and deep-fried potato products at 5.2 and 6.8, respectively. In conclusion, the TFA content of foods available on the German market is generally declining, but substantial variations are present. The t9/t11 index could be used as an indicator to determine ruminant fat. Practical applications: A number of studies provide evidence that a high TFA intake, particularly of industrial origin, adversely affects human health. The TFA content of foods could be reduced due to the introduction of several mandatory regulations and modifications regarding the hydrogenation process of oils. The most abundant dietary TFA are the isomers of trans C18:1. Unfortunately, the differentiation of these isomers is not yet very common, though the trans C18:1 profile differs depending on its origin (bacterial hydrogenation in the rumen or industrial hydrogenation). To date, data for TFA content

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb.

PubMed

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-05-27

Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb

PubMed Central

Kuzmina, Alona; Krasnopolsky, Simona; Taube, Ran

2017-01-01

ABSTRACT Early work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear. Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter. We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb. PMID:28340332

Effect of Trans, Trans-Farnesol on Pseudogymnoascus destructans and Several Closely Related Species.

PubMed

Raudabaugh, Daniel B; Miller, Andrew N

2015-12-01

Bat white-nose syndrome, caused by the psychrophilic fungus Pseudogymnoascus destructans, has dramatically reduced the populations of many hibernating North American bat species. The search for effective biological control agents targeting P. destructans is of great importance. We report that the sesquiterpene trans, trans-farnesol, which is also a Candida albicans quorum sensing compound, prevented in vitro conidial germination for at least 14 days and inhibited growth of preexisting hyphae of five P. destructans isolates in filtered potato dextrose broth at 10 °C. Depending on the inoculation concentrations, both spore and hyphal inhibition occurred upon exposure to concentrations as low as 15-20 µM trans, trans-farnesol. In contrast, most North American Pseudogymnoascus isolates were more tolerant to the exposure of trans, trans-farnesol. Our results suggest that some Candida isolates may have the potential to inhibit the growth of P. destructans and that the sesquiterpene trans, trans-farnesol has the potential to be utilized as a biological control agent.

Peptide conjugated magnetic nanoparticles for magnetically mediated energy delivery to lung cancer cells.

PubMed

Hauser, Anastasia K; Anderson, Kimberly W; Hilt, J Zach

2016-07-01

In the present study, we examine the effects of internalized peptide-conjugated iron oxide nanoparticles and their ability to locally convert alternating magnetic field (AMF) energy into other forms of energy (e.g., heat and rotational work). Dextran-coated iron oxide nanoparticles were functionalized with a cell penetrating peptide and after internalization by A549 and H358 cells were activated by an AMF. TAT-functionalized nanoparticles and AMF exposure increased reactive oxygen species generation compared with the nanoparticle system alone. The TAT-functionalized nanoparticles induced lysosomal membrane permeability and mitochondrial membrane depolarization, but these effects were not further enhanced by AMF treatment. Although not statistically significant, there are trends suggesting an increase in apoptosis via the Caspase 3/7 pathways when cells are exposed to TAT-functionalized nanoparticles combined with AMF. Our results indicate that internalized TAT-functionalized iron oxide nanoparticles activated by an AMF elicit cellular responses without a measurable temperature rise.

Peptide conjugated magnetic nanoparticles for magnetically mediated energy delivery to lung cancer cells

PubMed Central

Hauser, Anastasia K; Anderson, Kimberly W; Hilt, J Zach

2016-01-01

Aim: In the present study, we examine the effects of internalized peptide-conjugated iron oxide nanoparticles and their ability to locally convert alternating magnetic field (AMF) energy into other forms of energy (e.g., heat and rotational work). Materials & methods: Dextran-coated iron oxide nanoparticles were functionalized with a cell penetrating peptide and after internalization by A549 and H358 cells were activated by an AMF. Results: TAT-functionalized nanoparticles and AMF exposure increased reactive oxygen species generation compared with the nanoparticle system alone. The TAT-functionalized nanoparticles induced lysosomal membrane permeability and mitochondrial membrane depolarization, but these effects were not further enhanced by AMF treatment. Although not statistically significant, there are trends suggesting an increase in apoptosis via the Caspase 3/7 pathways when cells are exposed to TAT-functionalized nanoparticles combined with AMF. Conclusion: Our results indicate that internalized TAT-functionalized iron oxide nanoparticles activated by an AMF elicit cellular responses without a measurable temperature rise. PMID:27388639

Assessment and comparison of in vitro immunoregulatory activity of three astaxanthin stereoisomers

NASA Astrophysics Data System (ADS)

Sun, Weihong; Xing, Lihong; Lin, Hong; Leng, Kailiang; Zhai, Yuxiu; Liu, Xiaofang

2016-04-01

In recent years, the immune-modulatory role of all- trans astaxanthin from different pigment sources has been studied. It was reported that all- trans astaxanthin might exist as three stereoisomers, and the composition of all- trans stereoisomers in natural materials differs from that of synthetic products. However, the different biological effects of various all- trans stereoisomers still remain unclear. In the present study, we evaluated the bioactivity of three astaxanthin stereoisomers, ( 3S, 3'S)- trans-, ( 3R,3'R)- trans-and meso-trans-astaxanthin, in regulating cell-mediated immune response using mice lymphocytes and peritoneal exudates cells (PECs) systems. After the treatment with three astaxanthin stereoisomers (20 μmol L-1), the lymphocyte proliferation capacity, neutral red phagocytosis of PECs and natural killer (NK) cell cytotoxic activity were comparatively assessed. The results showed that all three astaxanthin stereoisomers significantly promoted lymphocyte proliferation, phagocytic capacity of PECs, and cytotoxic activity of NK cells. Moreover, the ( 3S,3'S)-trans-astaxanthin exhibited a much higher response than others.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

PubMed Central

Bao, Xingfeng; Zheng, Wanjun; Sugi, Naoko Hata; Agarwala, Kishan L; Xu, Qunli; Wang, Zichun; Tendyke, Karen; Lee, Winnie; Parent, Lana; Li, Wei; Cheng, Hongsheng; Shen, Yongchun; Taylor, Noel; Dezso, Zoltan; Du, Hong; Kotake, Yoshihiko; Zhao, Nanding; Wang, John; Postema, Maarten; Woodall-Jappe, Mary; Takase, Yasutaka; Uenaka, Toshimitsu; Kingston, David G I; Nomoto, Kenichi

2015-01-01

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment. PMID:25729885

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

PubMed Central

Gay, Darren C.; Wagner, Drew T.; Meinke, Jessica L.; Zogzas, Charles E.; Gay, Glen R.; Keatinge-Clay, Adrian T.

2016-01-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. PMID:26724270

Blocking GSK3β-mediated dynamin1 phosphorylation enhances BDNF-dependent TrkB endocytosis and the protective effects of BDNF in neuronal and mouse models of Alzheimer's disease.

PubMed

Liu, Xiang-Hua; Geng, Zhao; Yan, Jing; Li, Ting; Chen, Qun; Zhang, Qun-Ye; Chen, Zhe-Yu

2015-02-01

Endocytosis of tropomyosin related kinase B (TrkB) receptors has critical roles in brain-derived neurotrophic factor (BDNF) mediated signal transduction and biological function, however the mechanism that is governing TrkB endocytosis is still not completely understood. In this study, we showed that GSK3β, a key kinase in neuronal development and survival, could regulate TrkB endocytosis through phosphorylating dynamin1 (Dyn1) but not dynamin2 (Dyn2). Moreover, we found that beta-amyloid (Aβ) oligomer exposure could impair BDNF-dependent TrkB endocytosis and Akt activation through enhancing GSK3β activity in cultured hippocampal neurons, which suggested that BDNF-induced TrkB endocytosis and the subsequent signaling were impaired in neuronal model of Alzheimer's disease (AD). Notably, we found that inhibiting GSK3β phosphorylating Dyn1 by using TAT-Dyn1SpS could rescue the impaired TrkB endocytosis and Akt activation upon BDNF stimuli under Aβ exposure. Finally, TAT-Dyn1SpS could facilitate BDNF-mediated neuronal survival and cognitive enhancement in mouse models of AD. These results clarified a role of GSK3β in BDNF-dependent TrkB endocytosis and the subsequent signaling, and provided a potential new strategy by inhibiting GSK3β-induced Dyn1 phosphorylation for AD treatment. Copyright © 2014 Elsevier Inc. All rights reserved.

Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

PubMed Central

Kliewer, S; Garcia, J; Pearson, L; Soultanakis, E; Dasgupta, A; Gaynor, R

1989-01-01

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction. Images PMID:2529378

SPLICEFINDER – A Fast and Easy Screening Method for Active Protein Trans-Splicing Positions

PubMed Central

Eppmann, Simone; Busche, Alena; Dikovskaya, Dina; Dötsch, Volker; Mootz, Henning D.

2013-01-01

Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins. PMID:24023792

Trans-anethole ameliorates obesity via induction of browning in white adipocytes and activation of brown adipocytes.

PubMed

Kang, Nam Hyeon; Mukherjee, Sulagna; Min, Taesun; Kang, Sun Chul; Yun, Jong Won

2018-05-24

To treat obesity, suppression of white adipose tissue (WAT) expansion and activation of brown adipose tissue (BAT) are considered as potential therapeutic targets. Recent advances have been made in the induction of brown fat-like adipocytes (beige) in WAT, which represents an attractive potential strategy for the management and treatment of obesity. Use of natural compounds for browning of white adipocytes can be considered as a safe and novel strategy against obesity. Here, we report that trans-anethole (TA), a flavoring substance present in the essential oils of various plants, alleviated high fat diet (HFD)-induced obesity in mice models via elevation of the expression of beige-specific genes such as Ppargc1α, Prdm16, Ucp1, Cd137, Cited1, Tbx1, and Trem26. TA also regulated lipid metabolism in white adipocytes via reduction of adipogenesis and lipogenesis as well as elevation of lipolysis and fat oxidation. Moreover, TA exhibited thermogenic activity by increasing mitochondrial biogenesis in white adipocytes and activating brown adipocytes. In addition, molecular docking analysis enabled us to successfully predict core proteins for fat browning such as β3-adrenergic receptor (β3-AR) and sirtuin1 (SIRT1) based on their low binding energy interactions with TA for promotion of regulatory mechanisms. Indeed, agonistic and antagonistic studies demonstrated that TA induced browning of 3T3-L1 adipocytes through activation of β3-AR as well as the AMPK-mediated SIRT1 pathway regulating PPARα and PGC-1α. In conclusion, TA possesses potential therapeutic implications for treatment of obesity by playing multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, and promotion of lipid catabolism. Copyright © 2018. Published by Elsevier B.V.

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

Relationship between SU Subdomains That Regulate the Receptor-Mediated Transition from the Native (Fusion-Inhibited) to the Fusion-Active Conformation of the Murine Leukemia Virus Glycoprotein

PubMed Central

Lavillette, Dimitri; Ruggieri, Alessia; Boson, Bertrand; Maurice, Marielle; Cosset, François-Loïc

2002-01-01

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

PubMed

Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang

2014-11-03

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trans Fatty Acids

NASA Astrophysics Data System (ADS)

Doyle, Ellin

1997-09-01

Fats and their various fatty acid components seem to be a perennial concern of nutritionists and persons concerned with healthful diets. Advice on the consumption of saturated, polyunsaturated, monounsaturated, and total fat bombards us from magazines and newspapers. One of the newer players in this field is the group of trans fatty acids found predominantly in partially hydrogenated fats such as margarines and cooking fats. The controversy concerning dietary trans fatty acids was recently addressed in an American Heart Association (AHA) science advisory (1) and in a position paper from the American Society of Clinical Nutrition/American Institute of Nutrition (ASCN/AIN) (2). Both reports emphasize that the best preventive strategy for reducing risk for cardiovascular disease and some types of cancer is a reduction in total and saturated fats in the diet, but a reduction in the intake of trans fatty acids was also recommended. Although the actual health effects of trans fatty acids remain uncertain, experimental evidence indicates that consumption of trans fatty acids adversely affects serum lipid levels. Since elevated levels of serum cholesterol and triacylglycerols are associated with increased risk of cardiovascular disease, it follows that intake of trans fatty acids should be minimized.

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.

Mini-Review: Ergothioneine and Ovothiol Biosyntheses, an Unprecedented Trans-Sulfur Strategy in Natural Product Biosynthesis.

PubMed

Naowarojna, Nathchar; Cheng, Ronghai; Chen, Li; Quill, Melissa; Xu, Meiling; Zhao, Changming; Liu, Pinghua

2018-06-19

As one of the most abundant elements on earth, sulfur is part of many small molecular metabolites and is key to their biological activities. Over the past few decades, some general strategies have been discovered for the incorporation of sulfur into natural products. In this review, we summarize recent efforts in elucidating the biosynthetic details for two sulfur-containing metabolites, ergothioneine and ovothiol. Their biosyntheses involve an unprecedented trans-sulfur strategy, a combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation reaction and a PLP enzyme-mediated C-S lyase reaction.

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.

Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

PubMed

van der Gracht, Anouk M F; de Geus, Mark A R; Camps, Marcel G M; Ruckwardt, Tracy J; Sarris, Alexi J C; Bremmers, Jessica; Maurits, Elmer; Pawlak, Joanna B; Posthoorn, Michelle M; Bonger, Kimberly M; Filippov, Dmitri V; Overkleeft, Herman S; Robillard, Marc S; Ossendorp, Ferry; van Kasteren, Sander I

2018-06-15

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

Trans-National Scale-Up of Services in Global Health

PubMed Central

Shahin, Ilan; Sohal, Raman; Ginther, John; Hayden, Leigh; MacDonald, John A.; Mossman, Kathryn; Parikh, Himanshu; McGahan, Anita; Mitchell, Will; Bhattacharyya, Onil

2014-01-01

Background Scaling up innovative healthcare programs offers a means to improve access, quality, and health equity across multiple health areas. Despite large numbers of promising projects, little is known about successful efforts to scale up. This study examines trans-national scale, whereby a program operates in two or more countries. Trans-national scale is a distinct measure that reflects opportunities to replicate healthcare programs in multiple countries, thereby providing services to broader populations. Methods Based on the Center for Health Market Innovations (CHMI) database of nearly 1200 health programs, the study contrasts 116 programs that have achieved trans-national scale with 1,068 single-country programs. Data was collected on the programs' health focus, service activity, legal status, and funding sources, as well as the programs' locations (rural v. urban emphasis), and founding year; differences are reported with statistical significance. Findings This analysis examines 116 programs that have achieved trans-national scale (TNS) across multiple disease areas and activity types. Compared to 1,068 single-country programs, we find that trans-nationally scaled programs are more donor-reliant; more likely to focus on targeted health needs such as HIV/AIDS, TB, malaria, or family planning rather than provide more comprehensive general care; and more likely to engage in activities that support healthcare services rather than provide direct clinical care. Conclusion This work, based on a large data set of health programs, reports on trans-national scale with comparison to single-country programs. The work is a step towards understanding when programs are able to replicate their services as they attempt to expand health services for the poor across countries and health areas. A subset of these programs should be the subject of case studies to understand factors that affect the scaling process, particularly seeking to identify mechanisms that lead to

Metacognitions as Mediators of Gender Identity-related Anxiety.

PubMed

Fernie, Bruce A; Wright, Talen; Caselli, Gabriele; Nikčević, Ana V; Spada, Marcantonio M

2017-01-01

Research has found that the prevalence of psychological distress is substantially higher in transgender compared to cisgender populations. This study explored the role of metacognitions as mediators of anxiety in a sample comprising of cisgender and transgender individuals. One-hundred and twenty-five individuals (19 trans-male; 24 male; 25 trans-female; 57 female) completed a series of measures that assessed metacognitions, worry and anxiety. Correlation analyses were used to identify potential mediators of the relationship between gender identity and anxiety. A mediation model indicated that beliefs about thoughts concerning uncontrollability and danger entirely mediated the relationship between gender identity and anxiety (b = 2.00, bias corrected and accelerated confidence interval [0.68, 3.49]). Metacognitions play an important role in anxiety in transgender individuals. -Metacognitions were found to mediate anxiety in transgender and cisgender individuals. -The exploration of metacognitions in transgender individuals experiencing psychological distress may have clinical utility. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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

Supraspinal and spinal effects of L-trans-PDC, an inhibitor of glutamate transporter, on the micturition reflex in rats.

PubMed

Honda, Masashi; Yoshimura, Naoki; Hikita, Katsuya; Hinata, Nobuyuki; Muraoka, Kuniyasu; Saito, Motoaki; Chancellor, Michael B; Takenaka, Atsushi

2013-09-01

Glutamate is a major excitatory transmitter in the central nervous system, controlling lower urinary tract function. Five types of glutamate transporters such as GLAST (EAAT1), GLT-1 (EAAT2), EAAC-1 (EAAT3), EAAT4, and EAAT5 have been cloned so far. In the current study we tested whether L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), a non-selective inhibitor of glutamate transporters that increases endogenous glutamate concentration, can affect the micturition reflex in urethane anesthetized rats. Continuous cystometrograms (CMG, 0.04 ml/min infusion rate) were performed in two groups of urethane-anesthetized rats. A group of 18 rats was used for intrathecal administration of 1-10 µg of L-trans-PDC via an intrathecal catheter. In the second group of 18 rats, 1-10 µg of L-trans-PDC were administered intracerebroventricularly via a catheter inserted into the lateral ventricle. Micturition parameters were recorded and compared before and after drug administration. Intrathecal administration of L-trans-PDC at 1, 3, and 10 µg (n = 6 per dose) increased intercontraction intervals in dose dependent fashion, but did not affect postvoid residual or basal pressure at any doses tested. Intracerebroventricular administration of L-trans-PDC at 1, 3, and 10 µg (n = 6 per dose) also increased intercontraction intervals in dose dependent fashion, but did not affect postvoid residual or basal pressure at any doses tested. The current results show that, in urethane-anesthetized rats, suppression of glutamate transporters by L-trans-PDC has an inhibitory effect on the micturition reflex at supraspinal and spinal sites, possibly via activation of glutamate-mediated inhibitory pathways. Copyright © 2012 Wiley Periodicals, Inc.

All-trans retinoyl beta-glucose: chemical synthesis, growth-promoting activity, and metabolism in the rat.

PubMed

Barua, A B; Olson, J A

1991-01-01

All-trans retinoyl beta-glucose was chemically synthesized in good yield by reaction of retinoyl fluoride with glucose. Retinoyl glucose, which is soluble in water, shows growth-promoting activity similar to retinyl acetate in vitamin A-deficient rats. In metabolic studies, retinoyl glucose was found to be hydrolyzed to retinoic acid, but at a slower rate. The possible therapeutic uses of retinoyl glucose are discussed.

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain.

PubMed

Miyake, Zenshi; Takekawa, Mutsuhiro; Ge, Qingyuan; Saito, Haruo

2007-04-01

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.

Ligand-controlled, norbornene-mediated, regio- and diastereoselective rhodium-catalyzed intramolecular alkene hydrosilylation reactions.

PubMed

Hua, Yuanda; Nguyen, Hiep H; Scaggs, William R; Jeon, Junha

2013-07-05

Ligand-controlled, norbornene-mediated, regio- and diastereoselective rhodium-catalyzed intramolecular alkene hydrosilylation of homoallyl silyl ethers (1) exploiting either BINAP or 1,6-bis(diphenylphosphino)hexane (dpph) has been developed. This method permits selective access to either trans-oxasilacyclopentanes (trans-2) or oxasilacyclohexanes (3) at will. A substoichiometric amount of norbornene markedly increased both yield and selectivity. A norbornene-mediated hydride shuttle process is discussed.

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

PubMed Central

Ramsden, Christopher E.; Domenichiello, Anthony F.; Yuan, Zhi-Xin; Sapio, Matthew R.; Keyes, Gregory S.; Mishra, Santosh K.; Gross, Jacklyn R.; Majchrzak-Hong, Sharon; Zamora, Daisy; Horowitz, Mark S.; Davis, John M.; Sorokin, Alexander V.; Dey, Amit; LaPaglia, Danielle M.; Wheeler, Joshua J.; Vasko, Michael R.; Mehta, Nehal N.; Mannes, Andrew J.; Iadarola, Michael J.

2018-01-01

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy-or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene–related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber–mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch. PMID:28831021

Building coalitions: The interconnections between feminism and trans* activism in Spain.

PubMed

Lucas Platero, R; Ortega-Arjonilla, Esther

2016-01-01

What made current Spanish feminism shift toward transfeminism? Based on in-depth interviews and literature reviews, we explore what factors facilitated the participation of trans* women in Spanish feminism. Tracing the history through relevant events such as the National Feminist Conferences, it becomes clear that trans* women participated in the 1993, 2000, and 2009 conferences, posing relevant issues regarding prostitution, transgenderism, and the political subject of feminism. Our research allows a break with global oppositional narratives, in which these movements are in conflict, and highlights the importance of understanding the vernacular nuances that take place in a particular geopolitical context.

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

Insecticidal activity of basil oil, trans-anethole, estragole, and linalool to adult fruit flies of Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae.

PubMed

Chang, Chiou Ling; Cho, Il Kyu; Li, Qing X

2009-02-01

Basil oil and its three major active constituents (trans-anethole, estragole, and linalool) obtained from basil (Oscimum basilicum L.) were tested on three tephritid fruit fly species [Ceratitis capitata (Wiedemann), Bactrocera dorsalis (Hendel), and Bactrocera cucurbitae (Coquillett)] for insecticidal activity. All test chemicals acted fast and showed a steep dose-response relationship. The lethal times for 90% mortality/knockdown (LT90) of the three fly species to 10% of the test chemicals were between 8 and 38 min. The toxic action of basil oil in C. capitata occurred significantly faster than in B. cucurbitae but slightly faster than in B. dorsalis. Estragole acted faster in B. dorsalis than in C. capitata and B. cucurbitae. Linalool action was faster in B. dorsalis and C. capitata than in B. cucurbitae. trans-Anethole action was similar to all three species. Methyl eugenol acted faster in C. capitata and B. cucurbitae than in B. dorsalis. When linalool was mixed with cuelure (attractant to B. cucurbitae male), its potency to the three fly species decreased as the concentration of cuelure increased. This was due to linalool hydrolysis catalyzed by acetic acid from cuelure degradation, which was confirmed by chemical analysis. When methyl eugenol (B. dorsalis male attractant) was mixed with basil oil, trans-anethole, estragole, or linalool, it did not affect the toxicity of basil oil and linalool to B. dorsalis, but it did significantly decrease the toxicity of trans-anethole and estragole. Structural similarity between methyl eugenol and trans-anethole and estragole suggests that methyl eugenol might act at a site similar to that of trans-anethole and estragole and serve as an antagonist if an action site exists. Methyl eugenol also may play a physiological role on the toxicity reduction.

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

PubMed

Gay, Darren C; Wagner, Drew T; Meinke, Jessica L; Zogzas, Charles E; Gay, Glen R; Keatinge-Clay, Adrian T

2016-03-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of the influence of mediator complex in HIV-1 transcription.

PubMed

Ruiz, Alba; Pauls, Eduardo; Badia, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2014-10-03

HIV-1 exploits multiple host proteins during infection. siRNA-based screenings have identified new proteins implicated in different pathways of the viral cycle that participate in a broad range of cellular functions. The human Mediator complex (MED) is composed of 28 elements and represents a fundamental component of the transcription machinery, interacting with the RNA polymerase II enzyme and regulating its ability to express genes. Here, we provide an evaluation of the MED activity on HIV replication. Knockdown of 9 out of 28 human MED proteins significantly impaired viral replication without affecting cell viability, including MED6, MED7, MED11, MED14, MED21, MED26, MED27, MED28, and MED30. Impairment of viral replication by MED subunits was at a post-integration step. Inhibition of early HIV transcripts was observed by siRNA-mediated knockdown of MED6, MED7, MED11, MED14, and MED28, specifically affecting the transcription of the nascent viral mRNA transactivation-responsive element. In addition, MED14 and MED30 were shown to have special relevance during the formation of unspliced viral transcripts (p < 0.0005). Knockdown of the selected MED factors compromised HIV transcription induced by Tat, with the strongest inhibitory effect shown by siMED6 and siMED14 cells. Co-immunoprecipitation experiments suggested physical interaction between MED14 and HIV-1 Tat protein. A better understanding of the mechanisms and factors controlling HIV-1 transcription is key to addressing the development of new strategies required to inhibit HIV replication or reactivate HIV-1 from the latent reservoirs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Teratogenicity of isotretinoin revisited: species variation and the role of all-trans-retinoic acid.

PubMed

Nau, H

2001-11-01

This paper reviews the teratogenicity of isotretinoin in regard to aspects of species variation, toxicokinetics, and metabolism. Particular emphasis is given to the hypothesis that most effects of isotretinoin (13-cis-retinoic acid) are mediated by isomerization to the all-trans-retinoic acid. This mechanism of action would provide a basis for the understanding of species differences and the extrapolation of experimental results to the human situation and thus improve drug development. The insensitive species (rat, mouse) eliminate the drug rapidly through detoxification to the beta-glucuronide; also, placental transfer is limited in these species. On the other hand, in sensitive species (primates), the drug is predominantly metabolized to the active 13-cis-4-oxo-retinoic acid; placental transfer is more extensive here. The beta-glucuronides showed limited placental transfer in all species examined; these metabolites exhibited very low, if any, measurable concentrations in the human. The 13-cis-retinoic acid is not appreciably bound to cellular retinoid-binding proteins or nuclear receptors and exhibits low tissue distribution and placental transfer. Its access to the nucleus may be extensive. Because of the long half life of 13-cis-retinoic acid, continuous isomerization results in significant area under the concentration-time curve levels of all-trans-retinoic acid in the mouse, monkey and the human; the all-trans-retinoic acid formed is extensively distributed across the placenta and may be an important factor that contributes to the teratogenic potency of 13-cis-retinoic acid. Isomerization cannot explain the teratogenic effects of 13-cis-retinoic acid in the rat and rabbit. It is concluded that the high teratogenic activity of isotretinoin in sensitive species (human, monkey) is related to slow elimination of the 13-cis-isomer, to metabolism to the 4-oxo-derivative, to increased placental transfer, to continuous isomerization and significant exposure of the

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

PubMed

Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

Biotransformation of trans-1-chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd)

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

Schmidt, Tobias; Bertermann, Rüdiger; Rusch, George M.

2013-05-01

trans-1-Chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd) is a novel foam blowing and precision cleaning agent with a very low impact for global warming and ozone depletion. trans-HCFO-1233zd also has a low potential for toxicity in rodents and is negative in genotoxicity testing. The biotransformation of trans-HCFO-1233zd and kinetics of metabolite excretion with urine were assessed in vitro and in animals after inhalation exposures. For in vitro characterization, liver microsomes from rats, rabbits and humans were incubated with trans-HCFO-1233zd. Male Sprague Dawley rats and female New Zealand White rabbits were exposed to 2,000, 5,000 and 10,000 ppm for 6 h and urine was collected formore » 48 h after the end of the exposure. Study specimens were analyzed for metabolites using {sup 19}F NMR, LC-MS/MS and GC/MS. S-(3,3,3-trifluoro-trans-propenyl)-glutathione was identified as predominant metabolite of trans-HCFO-1233zd in all microsomal incubation experiments in the presence of glutathione. Products of the oxidative biotransformation of trans-HCFO-1233zd were only minor metabolites when glutathione was present. In rats, both 3,3,3-trifluorolactic acid and N-acetyl-(3,3,3-trifluoro-trans-propenyl)-L-cysteine were observed as major urinary metabolites. 3,3,3-Trifluorolactic acid was not detected in the urine of rabbits. Quantitation showed rapid excretion of both metabolites in both species (t{sub 1/2} < 6 h) and the extent of biotransformation of trans-HCFO-1233zd was determined as approximately 0.01% of received dose in rabbits and approximately 0.002% in rats. trans-HCFO-1233zd undergoes both oxidative biotransformation and glutathione conjugation at very low rates. The low extent of biotransformation and the rapid excretion of metabolites formed are consistent with the very low potential for toxicity of trans-HCFO-1233zd in mammals. - Highlights: ► No lethality and clinical signs were observed. ► Glutathione S-transferase and cytochrome P-450 dependent

Structural Elucidation of cis / trans Dicaffeoylquinic Acid Photoisomerization Using Ion Mobility Spectrometry-Mass Spectrometry

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

Zheng, Xueyun; Renslow, Ryan S.; Makola, Mpho M.

Due to the recently uncovered health benefits and anti-HIV activities of dicaffeoylquinic acids (diCQAs), understanding their structures and functions is of great interest for drug discovery efforts. DiCQAs are analytically challenging to identify and quantify since they commonly exist as a diverse mixture of positional and geometric (cis/trans) isomers. In this work, we utilized ion mobility spectrometry coupled with mass spectrometry to separate the various isomers before and after UV irradiation. The experimental collision cross sections were then compared with theoretical structures to differentiate and identify the diCQA isomers. Our analyses found that naturally the diCQAs existed predominantly as trans/transmore » isomers, but after 3 h of UV irradiation, cis/cis, cis/trans, trans/cis, and trans/trans isomers were all present in the mixture. This is the first report of successful differentiation of cis/trans diCQA isomers individually, which shows the great promise of IMS coupled with theoretical calculations for determining the structure and activity relationships of different isomers in drug discovery studies.« less

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.

Epstein-Barr Virus Glycoprotein gB and gHgL Can Mediate Fusion and Entry in trans, and Heat Can Act as a Partial Surrogate for gHgL and Trigger a Conformational Change in gB

PubMed Central

Chesnokova, Liudmila S.; Ahuja, Munish K.

2014-01-01

ABSTRACT Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrin αvβ5, αvβ6, or αvβ8. Fusion with a B cell requires gHgL, gp42, and gB and is triggered by an interaction between gp42 and human leukocyte antigen class II. We report here that, like alpha- and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL, and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing the fusion of cells expressing only gB, and the proteolytic digestion pattern of gB in virions changes in the same way following the exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high-affinity interaction of gHgL with an integrin contributes to the activation energy required to cause the refolding of gB from a prefusion to a postfusion conformation. IMPORTANCE The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha- and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes, implicating interactions between the ectodomains of the proteins in the activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB, possibly representing the refolding of gB from its prefusion to its postfusion conformation. PMID:25142593

Transcription co-activator SAYP mediates the action of STAT activator

PubMed Central

Panov, Vladislav V.; Kuzmina, Julia L.; Doronin, Semen A.; Kopantseva, Marina R.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Vorobyeva, Nadezhda E.; Shidlovskii, Yulii V.

2012-01-01

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT. PMID:22123744

Transcription co-activator SAYP mediates the action of STAT activator.

PubMed

Panov, Vladislav V; Kuzmina, Julia L; Doronin, Semen A; Kopantseva, Marina R; Nabirochkina, Elena N; Georgieva, Sofia G; Vorobyeva, Nadezhda E; Shidlovskii, Yulii V

2012-03-01

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

In vitro modeling of HIV proviral activity in microglia.

PubMed

Campbell, Lee A; Richie, Christopher T; Zhang, Yajun; Heathward, Emily J; Coke, Lamarque M; Park, Emily Y; Harvey, Brandon K

2017-12-01

Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll-like receptor-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

All-trans-retinoic acid inhibition of transforming growth factor-β-induced collagen gel contraction mediated by human Tenon fibroblasts: role of matrix metalloproteinases.

PubMed

Liu, Yang; Kimura, Kazuhiro; Orita, Tomoko; Teranishi, Shinichiro; Suzuki, Katsuyoshi; Sonoda, Koh-Hei

2015-04-01

Scarring and contraction of the conjunctiva are common complications of many ocular diseases. We investigated the effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon's capsule fibroblasts (HTFs) cultured in a three-dimensional collagen gel. HTFs were cultured in a three-dimensional gel of type I collagen and in the absence or presence of transforming growth factor (TGF)-β, ATRA, or an inhibitor of matrix metalloproteinases (MMPs). Collagen gel contraction was evaluated by measurement of gel diameter. The release of MMPs and tissue inhibitors of metalloproteinases (TIMPs) into culture supernatants was assessed by immunoblot analysis and gelatin zymography. The release of lactate dehydrogenase activity from HTFs was measured with a colorimetric assay kit. ATRA inhibited TGF-β-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. TGF-β induced the release of MMP-1, MMP-2 and MMP-3 by HTFs, and ATRA inhibited these effects of TGF-β on MMP-1 and MMP-3 release. ATRA also stimulated TIMP-1 release from HTFs in the presence of TGF-β. Furthermore, TGF-β-induced collagen gel contraction was blocked by the MMP inhibitor GM6001. ATRA did not exhibit cytotoxicity for HTFs. ATRA inhibited TGF-β-induced collagen gel contraction mediated by HTFs, likely in part by attenuating the production of MMP-1 and MMP-3 and by stimulating the production of TIMP-1. ATRA may therefore prove to be of clinical value for inhibition of scar formation in the conjunctiva. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Survey of the trans-resveratrol and trans-piceid content of cocoa-containing and chocolate products.

PubMed

Hurst, W Jeffrey; Glinski, Jan A; Miller, Kenneth B; Apgar, Joan; Davey, Matthew H; Stuart, David A

2008-09-24

Dietary resveratrol (3,4',5-trihydroxystilbene) has been implicated in the health benefits associated with grapes and red wine, more specifically with potential benefits for metabolic syndrome, energy use, and increased endurance. Levels of trans-resveratrol and its glucoside, trans-piceid, were determined in 19 top selling commercially available cocoa-containing and chocolate products from the U.S. market. Amounts of trans-resveratrol and trans-piceid were closely correlated with the amount of nonfat cocoa solids (NFCS) in the cocoa-containing products. Among these products, trans-resveratrol levels were highest in cocoa powders (1.85 +/- 0.43 microg/g), followed by unsweetened baking chocolates (1.24 +/- 0.22), semisweet chocolate baking chips (0.52 +/- 0.14), dark chocolates (0.35 +/- 0.08), milk chocolates (0.10 +/- 0.05), and chocolate syrups (0.09 +/- 0.02). These cocoa-containing and chocolate products have about 3-5 times more trans-piceid than trans-resveratrol. Levels of trans-piceid were highest in the cocoa powders (7.14 +/- 0.80 microg/g), followed by unsweetened baking chocolates (4.04 +/- 0.14), semisweet chocolate baking chips (2.01 +/- 0.18), dark chocolates (1.82 +/- 0.36), milk chocolates (0.44 +/- 0.06), and chocolate syrups (0.35 +/- 0.06). On an equal weight basis, cocoa powder had about half as much trans-resveratrol as the average California red wine. On a per serving basis, cocoa-containing and chocolate products had less trans-resveratrol than red wine and grape juice but more than roasted peanuts. Overall, these cocoa-containing and chocolate products rank second after red wines and grape juice in foods with the highest levels of total trans-resveratrol in the diet.

Polyoxometalate active charge-transfer material for mediated redox flow battery

DOEpatents

Anderson, Travis Mark; Hudak, Nicholas; Staiger, Chad; Pratt, Harry

2017-01-17

Redox flow batteries including a half-cell electrode chamber coupled to a current collecting electrode are disclosed herein. In a general embodiment, a separator is coupled to the half-cell electrode chamber. The half-cell electrode chamber comprises a first redox-active mediator and a second redox-active mediator. The first redox-active mediator and the second redox-active mediator are circulated through the half-cell electrode chamber into an external container. The container includes an active charge-transfer material. The active charge-transfer material has a redox potential between a redox potential of the first redox-active mediator and a redox potential of the second redox-active mediator. The active charge-transfer material is a polyoxometalate or derivative thereof. The redox flow battery may be particularly useful in energy storage solutions for renewable energy sources and for providing sustained power to an electrical grid.

Activation of MTK1/MEKK4 by GADD45 through Induced N-C Dissociation and Dimerization-Mediated trans Autophosphorylation of the MTK1 Kinase Domain▿ †

PubMed Central

Miyake, Zenshi; Takekawa, Mutsuhiro; Ge, Qingyuan; Saito, Haruo

2007-01-01

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45α/β/γ). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45. PMID:17242196

Fungal mediator tail subunits contain classical transcriptional activation domains.

PubMed

Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

2008-08-01

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

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.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

PubMed

Kim, Ha Won; Chan, Qilin; Afton, Scott E; Caruso, Joseph A; Lai, Barry; Weintraub, Neal L; Qin, Zhenyu

2012-02-01

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

Insulin elicits a ROS-activated and an IP₃-dependent Ca²⁺ release, which both impinge on GLUT4 translocation.

PubMed

Contreras-Ferrat, Ariel; Llanos, Paola; Vásquez, César; Espinosa, Alejandra; Osorio-Fuentealba, César; Arias-Calderon, Manuel; Lavandero, Sergio; Klip, Amira; Hidalgo, Cecilia; Jaimovich, Enrique

2014-05-01

Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47(phox)-NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47(phox) knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca(2+) release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca(2+) channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca(2+) transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,β phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca(2+) release and IP3-receptor-mediated mitochondrial Ca(2+) uptake, and that these signals jointly stimulate glucose uptake.

Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells.