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D E Levy

Publications and source records attributed to D E Levy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Physiological significance of STAT proteins: investigations through gene disruption in vivo.

Signal transducers and activators of transcription (STATs) were discovered as mediators of type I interferon-induced gene expression. This family of transcription factors has been found in widespread signaling pathways, especially those involving cytokines regulating the immune response. Because a plethora and often confusing set of activators for STAT proteins was observed in cell culture models, it became important to define the physiologically relevant actions of these molecules. One approach to this question has been through the targeted disruption of STAT genes in transgenic mice. Now that all seven STAT genes have been disrupted, both the high degree of STAT selectivity as well as many surprising and unexpected complexities are beginning to be characterized.

Animals↗
PubMed1999

Different mechanisms for the photoinduced production of oxidative DNA damage by fluoroquinolones differing in photostability.

Several fluoroquinolone antibacterial agents exhibit an adverse phototoxic effect in humans and are photo-cocarcinogenic in mice. The UV-induced production of reactive oxygen species plays a role in the toxicity and may be involved in carcinogenicity. Four fluoroquinolones were examined for the ability to photochemically produce oxidative damage in naked DNA. The major structural difference in the fluoroquinolones that would have an effect on their photostability is the functionality at the 8-position. At this position, 1-cyclopropyl-7-(2,8-diazbicyclo[4.3.0]non-8-yl)-6, 8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY y3118) contains a chlorine atom, lomefloxacin a fluorine atom, ciprofloxacin a proton, and moxifloxacin a methoxy group. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA was assessed by HPLC with electrochemical detection, and strand breaks were measured in pBR322 with agarose gel electrophoresis. The relative photolability of the fluoroquinolones correlated to the extent of production of 8-oxodGuo and strand breaks, with both UVA and UVB irradiation, in the following order: BAY y3118 approximately lomefloxacin > ciprofloxacin > moxifloxacin. Experiments were performed to determine whether the mechanism of damage was due to a type I (radical) or type II (singlet oxygen) pathway. Nitrogen depletion of oxygen resulted in a decrease in the extent of formation of 8-oxodGuo, suggesting that oxygen was involved. The use of selective radical or singlet oxygen inhibitors was inconclusive with respect to which pathway was involved. The use of D(2)O as a solvent, which would extend the lifetime of singlet oxygen, suggested that this species is involved in the formation of 8-oxodGuo by moxifloxacin and ciprofloxacin, but not by lomefloxacin and BAY y3118. Similarly, it was found that singlet oxygen was not involved in strand break formation. Thus, the evidence suggests that fluoroquinolones can photochemically produce DNA damage by both type I and type II mechanisms.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

The immune response modifier imiquimod requires STAT-1 for induction of interferon, interferon-stimulated genes, and interleukin-6.

Imiquimod is an oral inducer of interferon (IFN) and several other proinflammatory cytokines and has been successfully used topically as an antiviral agent for the treatment of genital warts. We have investigated the molecular mechanisms by which imiquimod induces the expression of IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in vivo, using mice deficient in various components of the IFN signaling system. Mice deficient in the transcription factor interferon regulatory factor 1 (IRF-1) or in the serine/threonine protein kinase PKR responded normally to imiquimod, producing high levels of circulating IFN and induction of several ISGs. On the other hand, when mice deficient in STAT-1 were treated, a 32-fold reduction in the level of circulating IFN was observed, together with a lack of induction of 2-5 oligo adenylate synthetase (2-5 OAS) and IRF-1 genes. Interestingly, there was also a lack of induction of interleukin-6 (IL-6) gene expression, although tumor necrosis factor was induced and readily detected in serum. In mice deficient in the type I IFN receptor, imiquimod induced levels of IFN similar to those in control mice, but again, neither 2-5 OAS, IRF-1, nor IL-6 genes were induced in mutant mice. Our results suggest that STAT-1 plays a critical role in the mechanism of gene activation by imiquimod. Moreover, induction of IL-6 gene expression appears to be dependent on components of the IFN signaling cascade.

Administration, Oral↗
PubMed1998

Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems.

The NS1 protein is the only nonstructural protein encoded by influenza A virus. It has been proposed that the NS1 performs several regulatory functions during the viral replication cycle, including the regulation of synthesis, transport, splicing, and translation of mRNAs. Through the use of reverse genetics, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated. Our results indicate that the NS1 of influenza A virus is an auxiliary (virulence) factor which plays a crucial role in inhibiting interferon-mediated antiviral responses of the host.

Animals↗
PubMed1998

Proto-oncogene PML controls genes devoted to MHC class I antigen presentation.

Fragments of foreign antigens associated with class I molecules of the major histocompatibility complex (MHC) are presented at the cell surface to elicit an immune response. This presentation requires the coordinated expression of several genes contained in the MHC, including those encoding the MHC class I heavy chain, the proteins LMP-2 and LMP-7, which are involved in the proteasomal degradation of cytosolic antigens into peptide fragments that are destined for association with MHC class I molecules, and TAP-1 and TAP-2, which transport these fragments across the membrane of the endoplasmic reticulum at the start of their journey to the cell surface. In many virus-transformed cell lines and spontaneous tumours, these genes are simultaneously repressed. However, the key factor(s) that are essential for their expression and repression have not been identified. Here we report that the proto-oncogene product PML induces expression of LMP-2, LMP-7, TAP-1 and TAP-2 in an MHC-class I-negative, recurrent tumour, leading to the re-expression of cell-surface MHC in tumours and to rejection of the tumours. PML also regulates MHC expression in untransformed fibroblasts. We conclude that malfunction of PML may enable a tumour to evade the immune defence of its host.

3T3 Cells↗
PubMed1998

Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7.

Interferon (IFN) genes are among the earliest transcriptional responses to virus infection of mammalian cells. Although the regulation of the IFNbeta gene has been well characterized, the induction of the large family of IFNalpha genes has remained obscure. We report that the IFNalpha genes can be divided into two groups: an immediate-early response gene (IFNalpha4) which is induced rapidly and without the need for ongoing protein synthesis; and a set of genes that display delayed induction, consisting of at least IFNalpha2, 5, 6 and 8, which are induced more slowly and require cellular protein synthesis. One protein that must be synthesized for induction of the delayed gene set is IFN itself, presumably IFNalpha4 or IFNbeta, which stimulates the Jak-Stat pathway through the IFN receptor, resulting in activation of the transcription factor interferon-stimulated gene factor 3 (ISGF3). Among the IFN-stimulated genes induced through this positive feedback loop is the IFN regulatory factor (IRF) protein, IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus-specific signals, involving two C-terminal serine residues, are required for induction of the delayed IFNalpha gene set.

Animals↗
PubMed1998

Superantigen-activated T cells redirected by a bispecific antibody inhibit vesicular stomatitis virus replication in vitro and in vivo.

A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo.

Animals↗
PubMed1998

Matrix metalloproteinase inhibitors: a structure-activity study.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

Chromatography, High Pressure Liquid↗
PubMed1998

Analysis of interferon-regulated proteins binding the interferon-alpha-stimulated response element.

Interferon mediates its biological effects of antiviral, antiproliferative, and immunomodulatory activities through induction of specific gene expression. Transcription of target genes is regulated through a set of transcription factors that bind to specific cis-acting regulatory sequences in target promoters and enhancers. The activity or abundance of these transcription factors is modulated by interferon treatment. Because of their DNA binding activity, regulatory factors can be recognized and characterized using methods to detect protein-DNA complexes. The availability of antibodies directed against common interferon regulatory proteins coupled with analysis of binding-site specificity provides comprehensive analysis.

Base Sequence↗
PubMed1998

The role of interferon in influenza virus tissue tropism.

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.

Administration, Intranasal↗
PubMed1997

Regulation of interferon-alpha responsiveness by the duration of Janus kinase activity.

Daudi B lymphoblastoid cells are highly sensitive to the anti-growth and anti-viral effects of interferon (IFN). Unlike many cell lines, these cells show prolonged transcription of IFN-stimulated genes following treatment with IFN-alpha. This prolonged response correlated with the continued presence of the activated transcription factor, IFN-stimulated gene factor 3 (ISGF3). Pulse-chase labeling experiments indicated that the half-life of the phosphorylation of signal transducers and activators of transcription (Stat)1 and Stat2 was short (<2 h) although the turnover of the proteins themselves was slow (>24 h), indicative of a constitutive phosphatase activity. The administration of protein-tyrosine kinase inhibitors at any time point during IFN stimulation led to rapid inhibition of the response, indicating that tyrosine kinase activity was continuously required. Catalytic activity of Jak1 and Tyk2 kinases remained elevated for prolonged periods following stimulation. Continuous presence of IFN-alpha was necessary for maintaining prolonged activation of ISGF3 and of Janus kinases, an activity that was blocked by antibodies to IFN-alpha or by cycloheximide. Conditioned medium of IFN-alpha-stimulated cells was capable of stimulating STAT activation in naive cells. Taken together, these results suggest that the response to IFN-alpha is controlled by the duration of stimulated Janus kinase activity over the background of constitutive dephosphorylation and that this response can be sustained by autocrine secretion of IFN-alpha.

Antineoplastic Agents↗
PubMed1997

Structure of the hydrogen bonding complex of O6-methylguanine with cytosine and thymine during DNA replication.

During DNA replication, mutations occur when an incorrect dNTP is incorporated opposite a carcinogen-modified nucleotide. We have probed the structures of the interaction between O 6-methylguanine ( O 6mG) and cytosine and thymine during replication by kinetic means in order to examine the structure during the rate determining step. The kinetics of incorporation of dCTP and dTTP opposite O 6mG and three analogs, S 6-methyl-6-thioguanine, O 6-methyl-1-deazaguanine and O 6-methylhypoxanthine, have been measured with four polymerases, the Klenow fragment of DNA polymerase I, the Klenow fragment with the proof-reading exonuclease inactivated, Taq and Tth polymerases. In the insertion of dTTP opposite O 6mG, a large decrease in V max/ K m was observed only upon modification of the N1 position. This result is consistent with a Watson-Crick type configuration. For the incorporation of dCTP, the V max/ K m was significantly decreased only with removal of the exocyclic amino group at the 2 position. The pH dependence of the ratio of incorporation of dCTP and dTTP was independent of pH at physiological pH. This result suggests that dCTP is incorporated via an uncharged complex such as the wobble configuration.

Cytosine↗
PubMed1997

Stat2 is a transcriptional activator that requires sequence-specific contacts provided by stat1 and p48 for stable interaction with DNA.

Transcriptional responses to interferon (IFN) are mediated by tyrosine phosphorylation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (Stat) family. The Stat1 protein is required for all transcriptional responses to IFN (both type I and type II). Responses to type I IFN (alpha and beta) also require Stat2 and the IFN regulatory factor family protein p48, which form a heterotrimeric transcription complex with Stat1 termed ISGF3. Stat1 homodimers formed in response to IFN-gamma treatment can also interact with p48 and function as transcriptional activators. We now show that Stat2 is capable of forming a stable homodimer that interacts with p48, can be recruited to DNA, and can activate transcription, raising a question of why Stat1 is required. Analysis of the transcriptional competence, affinity, and specificity of Stat2-p48 complexes compared with other Stat protein-containing transcription factor complexes suggests distinct roles for each component. Although Stat2 is a potent transactivator, it does not interact stably with DNA in complex with p48 alone. Adding Stat1 increases the affinity and alters the sequence selectivity of p48-DNA interactions by contacting a half-site of its palindromic recognition motif adjacent to a p48 interaction sequence. Thus, ISGF3 assembly involves p48 functioning as an adaptor protein to recruit Stat1 and Stat2 to an IFN-alpha-stimulated response element, Stat2 contributes a potent transactivation domain but is unable to directly contact DNA, while Stat1 stabilizes the heteromeric complex by contacting DNA directly.

Biopolymers↗
PubMed1997

The house that Jak/Stat built.

An international workshop on "Cytokines, Signaling and the Jak/Stat Pathway' organized by Peter C. Heinrich of the Klinikum der RWTH Aachen under the auspices of DFG-Forschergruppe was held in Aachen, Germany, on 4-5 October 1996. In this review David Levy notes that even though some diagrams shown at the meeting displayed more arrows than there were meeting participants, remarkable progress has been made in the unraveling of the complexities of cytokine signal transduction. The central issue in the field of cytokine signaling is specificity, and this issue was addressed through experimental results from biochemical, molecular, and genetic studies of signal transduction pathways.

Amino Acid Sequence↗
PubMed1997

Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons.

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Acute-Phase Proteins↗
PubMed1996

Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease.

The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens.

Animals↗
PubMed1996

ISGF3 gamma p48, a specificity switch for interferon activated transcription factors.

Interferon (IFN) induces gene expression by phosphorylating latent transcription factors of the STAT family. Two different STAT multimeric complexes that bind distinct enhancer elements are activated by IFN alpha and IFN gamma, dictated by the DNA-binding protein ISGF3 gamma p48. This protein, a member of the IFN regulatory factor (IFR) family, acts as an adaptor protein to redirect STAT multimers from their intrinsic palindromic sequence specificity to interactions with a composite element composed of an IRF site juxtaposed with a STAT half-site. Sequence similarity within the IRF family suggests that other members could serve as adaptor proteins for transcriptional activators. Recent evidence suggests that PIP (LSIRF) sequesters the Ets protein PU.1 at a composite DNA element lends support to this adaptor hypothesis.

Amino Acid Sequence↗