Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), is being studied as a photosensitizer for virus inactivation in red blood cell concentrates (RBCC). The RBCC spiked with cell-free human immunodeficiency virus (HIV) or with HIV actively replicating in the T-lymphocytic cell line CEM can be successfully inactivated (> or = 6 log10) when exposed to 2 microM Pc 4 and 90 J/cm2 red light (600-800 nm). Inactivation of > or = 6 log10 inducible HIV in the latently infected promonocytic cell line U1 occurred at 22.5 J/cm2 (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996). In order to understand the reason for the increased susceptibility of U1 to photosensitized inactivation we looked for induction of apoptosis by photodynamic treatment (PDT). Agarose gel electrophoresis was used to observe the appearance of a characteristic 180-200 base pair DNA ladder, which can indicate apoptosis. Using this assay it is shown that Pc 4 treatment induced apoptosis in U1 cells in a light dose-dependent manner, starting 30 min after light exposure. Using the ApopTag Plus kit (which attaches a fluorescent label to the 3'-OH ends of the degraded DNA) and flow cytometry, the percentage of cells undergoing apoptosis was quantitated. At 10.5 J/cm2, 3 h after light exposure, about 92.5% of the cells were apoptotic. Under these conditions 99% of the cells eventually die. The CEM cells similarly treated underwent apoptosis at slower kinetics and required higher light doses. Other cell lines latently infected with HIV (ACH-2 and OM 10.1) were as sensitive as U1 to HIV inactivation by Pc 4-PDT (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996) and underwent apoptosis at a similar kinetic. These results suggest that the enhanced inactivation of HIV in latently infected cells compared to CEM cells by Pc 4-PDT may be due, at least in part, to apoptosis in the former.
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PMID:Silicon phthalocyanine Pc 4 and red light causes apoptosis in HIV-infected cells. 907 31

To develop a rapid and sensitive means of detecting cell-associated human immunodeficiency virus (HIV), donor cells from HIV seropositive patients were treated with the potent viral activator sodium-n-butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of RT-PCR was estimated to be equivalent to 1 x 10(-16) grams (0.1 fg) or approximately 64 copies of the input standard viral RNA per reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH-2 and U1 cell lines were used as representatives of T-lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT-PCR assays. When peripheral blood mononuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV-positive PBMCs by RT-PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell-associated viral load in vivo and without possible mutations brought about by long-term co-cultivation assays with cells from seronegative donors.
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PMID:Rapid and sensitive detection of cell-associated HIV-1 in latently infected cell lines and in patient cells using sodium-n-butyrate induction and RT-PCR. 917 66

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).
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PMID:Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1. 919 10

The immunodeficiency present in patients with lepromatous leprosy is characterized by the limited proliferation of T lymphocytes, and is explained in part by the impaired synthesis of interleukin-2 (IL-2). Diacylglycerol (DAG) and calcium produce the activation of PKC, ERK and JNK kinases, implying a normal IL-2 response. Phorbol esters, such as PMA, can substitute for DAG and are mitogenic to human T and B cells activating several cytokine-encoding genes. Ionophore A23187 increases calcium permeability across the cellular membrane to the cytosol of lymphoid cells and is considered a co-mitogen of T lymphocytes. Here we report that: 1) PHA-activated T lymphocytes from LL patients can be separated in vitro into two groups: a) responders (R) with a stimulation index (SI) of > 10 and (b) nonresponders (NR) with a SI of < 10. 2) The proliferative responses of cells from LL(R), LL(NR) and normal subjects were measured after being stimulated with: I, PHA, PMA, PMA + I PHA + PMA and PHA + PMA + ionophore (PPI). The most important result occurs in LL(NR) patients whose cells did not respond to PHA stimulation but increased to normal levels of proliferation when they were stimulated with PMA. Furthermore, the three groups, (NR, R and normals) strongly increased their responses when they were incubated with PPi. 3) Finally, Il-2 concentrations in the supernatants of cultures of T lymphocytes from LL(NR), LL(R) and controls were relatively low when they were incubated with PHA or PMA, but the addition of ionophore to PMA and the combination of PHA + PMA strongly increased the production of IL-2 in all of them, reaching the optimum IL-2 concentration when PPI is used. It can be concluded that the use of PMA, analogous to DAG, and ionophore A23187 (calcium increaser) in cultures of mitogen-activated T lymphocytes from LL patients induced the expression of the IL-2 gene, thus correcting the inadequate proliferation of T cells from LL patients.
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PMID:Effect of phorbol myristate acetate (PMA) and ionophore A23187 on interleukin-2 levels and proliferation of activated T lymphocytes from patients with lepromatous leprosy. 920 56

NMR and CD studies were carried out on a peptide representing the hydrophobic N-terminal domain of envelope glycoprotein of human immunodeficiency virus type-1 in solutions of varying polarity. It was found that in aquaeous solution the amide proton of glycine in the FLG motif resonated at a considerably high field and its chemical shift, within the limit of experimental precision, had a temperature coefficient of zero in the range studied. The upfield shift of NH of the glycine could be largely attributed to the ring-current effect of phenylalanine in the FLG motif that participated in a type-1 beta turn with a short Cbeta(i)-NH(i+2) distance. The slower proton-deuterion exchange for the glycine amide proton relative to that of other glycines was consistent with a folded structure for the motif in aquaeous solution. Results of the molecular simulation showed that this proton was shielded from the solvent by non-polar side chains of the amino acid residues surrounding the turn stabilized by hydrophobic interactions, thus explaining the zero temperature coefficient of the proton chemical shift. The structural stabilizing effect of the hydrophobic interaction was supported by the behavior of the proton in less polar Me2SO solution, in which the anomaly in the chemical shift and its temperature coefficient was less prominent. Detailed secondary-structure analysis suggested that the beta turn of the FLG motif may act as an initiation core for helix formation, probably because the turn readily transforms into helical form.
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PMID:The FLG motif in the N-terminal region of glucoprotein 41 of human immunodeficiency virus type 1 adopts a type-I beta turn in aqueous solution and serves as the initiation site for helix formation. 928 13

The role of the N-myristoylation of the human immunodeficiency virus type 1 (HIV-1) gag protein in ACH-2 cells was studied. The infectivity of HIV-1 from the cells stimulated with phorbol 12-myristate 13-acetate (PMA) was suppressed by pretreatment with N-myristoyl glycinal diethylacetal (N-Myr-GOA), a potent N-myristoylation inhibitor, and the blockage of myristoylation resulted in accumulation of immature gag precursors. The viral particles which budded from the non-N-Myr-GOA-treated ACH-2 cells stimulated with PMA exhibited a typical viral phenotype, whereas those which budded from the N-Myr-GOA-treated ACH-2 cells stimulated with PMA were twisted, as observed electron microscopically. In electron microscopic analyses with gold-labeled monoclonal antibodies to gag and env, gag and env were detected adjacent to each other in the PMA-stimulated ACH-2, but no env was detected in the cells treated with N-Myr-GOA. Taken together, the results suggest that the myristoylation of HIV-1 gag seems to be responsible for both maturation of gag and acquisition of HIV-1 infectivity.
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PMID:Blockage of N-myristoylation of HIV-1 gag induces the production of impotent progeny virus. 929 93

Nucleic acid isolation for amplification-based diagnostics requires techniques that do not co-purify inhibitors of DNA polymerases. Also, other requirements for an ideal sample preparation technology include ease of use, capability for automation, high recovery and the use of nontoxic reagents. Affinity purification techniques provide high purification factor with minimal sample processing. Hybridization is the affinity interaction specific to nucleic acids and thus provides a uniquely advantageous method for purifying DNA or RNA for subsequent manipulation. Nonionic (morpholino) probes (Neu-Probes, AntiVirals, Corvallis, OR, USA) have several unique hybridization properties, including resistance to nucleases and the ability to hybridize independently of salt concentration. Therefore, such probes provide advantages over DNA probes for sample preparation by hybridization capture. Three formats for hybridization-based purification of human immunodeficiency virus (HIV) RNA were evaluated using RNA transcripts spiked into crude lysates of normal human plasma. Indirect capture used streptavidin-coated microparticles to capture hybrids of biotinylated capture probes and HIV RNA. Direct capture used particles precoated with probes. In addition, a novel method for acceleration of sequence-specific hybridization was developed and shown to give consistently high recoveries.
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PMID:Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes. 942 46

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.
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PMID:The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. 942 62

Transition from latency to active replication is a crucial stage for the process of human immunodeficiency virus type 1 (HIV-1) infection and life cycle. HIV-1 replication in latently infected cells can be strongly induced by the cytokine tumor necrosis factor alpha (TNF-alpha) and the proliferation-arresting chemical sodium butyrate (NaB). We have investigated the ability of the drug 9-nitrocamptothecin (9NC), a potent cellular topoisomerase I (topo I) inhibitor currently in clinical trials in cancer patients, to regulate HIV-1 replication in latently infected lymphocytic ACH-2 cells on reactivation with either TNF-alpha or NaB. Treatment of ACH-2 cells with 9NC alone resulted in increased levels of viral transcripts, while there was a slight reduction or no change in the levels of host cell transcripts. However, pretreatment of ACH-2 cells with 9NC inhibited TNF-alpha-induced extracellular HIV-1 p24 levels up to approximately 95% and nearly 80% of the cell-associated viral RNAs. The quantitative decrease in viral products was concomitant with a decrease in cellular gene expression and induction of apoptosis in the host cells. 9NC blocked the infected cells at the boundary of the S and G2 phases, resulting in an accelerated apoptosis that was further enhanced with TNF-alpha treatment. Similar results were observed following concurrent exposure to TNF-alpha and 9NC, but 9NC failed to inhibit upregulation of HIV-1 mRNA in ACH-2 cells exposed to TNF-alpha before 9NC treatment. Further, 9NC had no inhibitory effect on NaB-induced apoptosis and upregulation of HIV-1 mRNA expression regardless of whether 9NC and NaB were used concurrently or in various treatment sequences. In uninfected lymphocytic CEM cells derived from a common parental cell line, a slight downregulation of cellular gene expression was detected along with low-level apoptosis. These results demonstrate that 9NC impairs TNF-alpha-induced, but not NaB-induced, HIV-1 activation, and suggest a means of inhibiting active HIV-1 viremia arising as a result of elevated TNF-alpha levels.
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PMID:9-Nitrocamptothecin inhibits tumor recrosis factor-mediated activation of human immunodeficiency virus type 1 and enhances apoptosis in a latently infected T cell clone. 945 50

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.
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PMID:Plasma membrane-targeted Raf kinase activates NF-kappaB and human immunodeficiency virus type 1 replication in T lymphocytes. 952 98


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