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D E Levy

Publications and source records attributed to D E Levy.

At least 19 recordsLinked to original sources
PubMed1992

Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins.

Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex. We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3 alpha.

Amino Acid Sequence↗
PubMed1992

Urgent therapy for stroke. Part I. Pilot study of tissue plasminogen activator administered within 90 minutes.

BACKGROUND AND PURPOSE: Thrombolytic agents hold theoretical promise as therapy for cerebral infarction. This study was designed to evaluate the safety of tissue plasminogen activator, to accomplish urgent patient treatment, and to estimate potential efficacy of tissue plasminogen activator. METHODS: Following neurological evaluation and computed tomography of the brain, patients with acute ischemic stroke were evaluated and treated with intravenous tissue plasminogen activator under an open-label, dose-escalation design within 90 minutes from symptom onset. End points examined included symptomatic and asymptomatic intracranial hematoma, systemic hemorrhage, and neurological outcome at 2 hours, 24 hours, and 3 months. RESULTS: Seventy-four patients were treated within 90 minutes of symptom onset over seven dose tiers of tissue plasminogen activator, ranging from 0.35 mg/kg to 1.08 mg/kg. Intracranial hematoma with associated neurological deterioration occurred in three patients and was related to increasing doses of tissue plasminogen activator (p = 0.045). Intracranial hematoma did not occur in any of the 58 patients treated with less than or equal to 0.85 mg/kg. Major neurological improvement occurred in 22 patients (30%) at 2 hours from the initiation of tissue plasminogen activator and in a total of 34 patients (46%) at 24 hours, but major neurological improvement was not related to increasing doses of tissue plasminogen activator or to stroke type. CONCLUSIONS: Patients with acute stroke can be evaluated and treated within 90 minutes. Tissue plasminogen activator for acute ischemic infarction is not without risk, but the potential for clinical benefit justifies a randomized clinical trial. To date, differences in hemorrhagic risk or neurological benefit of tissue plasminogen activator for particular ischemic stroke types are not apparent.

Aged↗
PubMed1992

Urgent therapy for stroke. Part II. Pilot study of tissue plasminogen activator administered 91-180 minutes from onset.

BACKGROUND AND PURPOSE: Renewed interest in thrombolytic therapy as potential treatment for patients with acute ischemic stroke prompted a dose-escalation safety study of tissue plasminogen activator in patients with very early (less than or equal to 90 minutes; see Part I) neurological symptoms. To test whether this stringent entry window might be safely lengthened, a second study was organized to test tissue plasminogen activator in patients with symptoms of 91-180 minutes' duration before treatment. METHODS: An open-label, dose-escalation design was chosen. Eligible patients had pretreatment head computerized tomographic scanning and treatment begun 91-180 minutes from stroke onset. End points examined included the incidence of symptomatic and asymptomatic intracranial hemorrhage, other bleeding, and clinical outcome at 2 hours, 24 hours, and 3 months after treatment. RESULTS: Twenty patients were treated at three hospitals in 13 months. Three doses were tested: 0.6 mg/kg (n = 8), 0.85 mg/kg (n = 6), and 0.95 mg/kg (n = 6). Two patients, one each at the two highest doses, sustained fatal intracerebral hemorrhages. Three patients (15%) improved by greater than or equal to 4 points on the National Institutes of Health Stroke Scale by 24 hours. CONCLUSIONS: These observations suggest that tissue plasminogen activator treatment of acute ischemic stroke 91-180 minutes from onset in doses of greater than or equal to 0.85 mg/kg is attended by a risk of intracerebral hemorrhage approximating 17% (range 3-44%, 95% confidence interval). The rate of early neurological improvement observed in this study was small but does not exclude an improvement over the natural history. Future study with placebo control subjects and stratification by time to treatment is indicated.

Aged↗
PubMed1991

Protein kinase activity required for an early step in interferon-alpha signaling.

Interferon-alpha (IFN alpha) induces an immediate transcriptional response of a restricted set of genes in target cells. Specific transcription is mediated by the cytoplasmic activation of a transcription factor complex termed ISGF3. ISGF3 is a multimeric protein complex composed of a regulatory component (ISGF3 alpha), which is activated following IFN alpha treatment, and a DNA-binding component (ISGF3 gamma), which recognizes the IFN alpha-stimulated response element (ISRE). Following activation, ISGF3 alpha translocates to the nucleus where ISGF3 assembles as a high affinity complex on the ISRE. The biochemical basis for receptor-mediated activation of ISGF3 is unknown. We report that two potent protein kinase inhibitors, staurosporine and K-252a, ablated the transcriptional response to IFN alpha treatment. These inhibitors prevented the activation of the ISGF3 alpha component without affecting the ISGF3 gamma component, resulting in no accumulation of mature ISGF3 in nuclei of treated cells. Although these agents are potent inhibitors of protein kinase C (PKC), PKC does not mediate ISGF3 alpha activation. Down-regulation of PKC by chronic exposure of cells to 12-O-tetradecanoylphorbol-13-acetate, which led to complete loss of PKC-immunoreactive material, failed to ablate the transcriptional response to IFN alpha or the activation of ISGF3 alpha. The PKC-specific inhibitor calphostin C did not perturb activation or nuclear accumulation of ISGF3. We conclude that a novel, staurosporine/K-252a-sensitive kinase is required for ISGF3 activity and may participate in receptor-mediated signal transduction.

Alkaloids↗
PubMed1991

Signal transduction pathway activating interferon-alpha-stimulated gene expression.

Interferon-alpha (IFN alpha) causes profound physiological changes following binding to susceptible target cells. These changes, which include induction of an antiviral state, inhibition of cellular proliferation, and modulation of differentiation, require the transcriptional activation of a set of genes. We have characterized the macromolecular components required for this stimulation of gene expression in order to define the biochemical mechanism of IFN alpha signal transduction. IFN alpha stimulated genes (ISGs) are immediate response genes which utilize a pre-existing set of proteins to mediate their induction. A 15 bp IFN alpha-inducible enhancer element present in the promoters of IFN alpha-stimulated genes, termed the IFN alpha stimulated response element (ISRE), is the genetic target for activation of ISGs. This DNA sequence is both necessary and sufficient for transcriptional activation and is the target for action of a positive transcription factor termed ISGF3. The active, DNA-binding form of ISGF3 is only found in cells which have been exposed to IFN alpha; however, it is activated from a silent form present in all responsive cells. ISGF3 is a multimeric complex assembled from cytoplasmic precursors which are translocated to the nucleus in response to IFN alpha. Assembly and translocation of ISGF3 is the earliest defined event in the IFN alpha response pathway.

Cytoplasm↗
PubMed1990

Synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor ISGF3.

Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1990

ISGF3, the transcriptional activator induced by interferon alpha, consists of multiple interacting polypeptide chains.

Interferon-stimulated gene factor 3 (ISGF3) is the ligand-dependent transcriptional activator that, in response to interferon treatment, is assembled in the cell cytoplasm, is translocated to the nucleus, and binds the consensus DNA site, the interferon-stimulated response element. We have purified ISGF3 and identified its constituent proteins: a DNA-binding protein of 48 kDa and three larger polypeptides (84, 91, and 113 kDa), which themselves do not have DNA-binding activity. The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription.

Base Sequence↗
PubMed1990

Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator.

The interaction of interferon-alpha (IFN-alpha) with a specific cell-surface receptor elicits physiological changes that rely on rapid transcriptional activation of a group of IFN-alpha-stimulated genes (ISGs). The IFN-stimulated response element (ISRE), a conserved regulatory element of all ISGs, is the target for transcriptional activation by the positive regulator IFN-stimulated gene factor-3 (ISGF3). We reported previously that post-translational activation of ISGF3 in the cytoplasm of IFN-alpha-treated cells requires two cytoplasmic activities (ISGF3 alpha and ISGF3 gamma) to produce an ISRE-binding complex that accumulates in the nucleus. In this study, we show that these activities are actually distinct subunits of the ISGF3 complex, which associate through noncovalent interaction. Sedimentation analysis, protein renaturation, and photoaffinity cross-linking of enriched preparations of cytoplasmic ISGF3 alpha and ISGF3 gamma and of nuclear ISGF3 demonstrated that ISGF3 gamma was a 48-kD polypeptide with intrinsic, low-affinity DNA-binding activity. Four polypeptides of 48, 84, 91, and 113 kD bound to the ISRE in vitro; the larger three polypeptides most likely compose the ISGF3 alpha component. These ISGF3 alpha polypeptides were unable to bind DNA alone but formed a DNA-binding complex in conjunction with ISGF3 gamma. The resulting heteromeric complex had the same ISRE-binding specificity as the individual ISGF3 gamma polypeptide but approximately 25-fold higher affinity. Whereas ISGF3 gamma partitioned between the cytoplasm and nucleus in unstimulated cells, ISGF3 alpha was stimulated to translocate to the nucleus only following IFN-alpha treatment, resulting in preferential nuclear accumulation of both ISGF3 alpha and ISGF3 gamma as a stable ISGF3-ISRE complex. This regulated nuclear translocation of an activated transcription factor subunit maintained the specificity and rapidity of the IFN-alpha signaling pathway.

Base Sequence↗
PubMed1979

Delayed pentobarbital administration limits ischemic brain damage in gerbils.

The capacity of delayed barbiturate administration to limit brain damage after unilateralcerebral ischemia was examined histologically in gerbils. The right common carotid artery was occluded in 50 animals under brief (3-minute) halothane anesthesia; 18 animals (36%) developed motor abnormalities consistent with stroke. The arterial clasps were removed after 1 hour and the abnormal animals were divided into treatment and placebo groups. Treated gerbils received sodium pentobarbital (70 mg/kg) intarperitoneally 1 hour after clasp removal and a smaller dose (50 mg/kg) 2 hours later; these animals lost corneal reflexes but retained spontaneous respiration and were kept normothermic. Animals in the placebo group received equivalent volumes of normal saline. Except for the period of anesthesia, both groups had similar postischemic motor behavior. Neuropathological examination of animals killed by perfusion-fixation after 24 hours revealed fewer pentobarbital-treated animals with shift of midline structures and with ipsilateral ischemic damage (including infarction). Compared with the placebo group, there was less extensive neuronal ischemic cell change in five regions of the ipsilateral cerebral hemispheres of the pentobarbital-treated animals (p less than 0.05). The results suggest that barbiturates administered as long as 1 hour after the end of an ischemic insult can still limit brain damage.

Animals↗
PubMed1979

Selective chromatolysis of neurons in the gerbil brain: a possible consequence of "epileptic" activity produced by common carotid artery occlusion.

Unilateral (50 to 118 minutes) and bilateral (2 to 33 minutes) carotid artery occlusion in gerbils resulted in two distinct types of neuronal alteration: ischemic cell change (ICC) in selectively vulnerable brain regions, and selective chromatolysis (SC) confined to the deeper layers of the cortex, the Sommer sector of zone h-1, and the paramedian region (PM) of the hippocampus. In typical SC the nucleus was eccentric and the Nissl substance was lost in the central eosinophilic cytoplasm. In electron micrographs this area of cytoplasm showed disruption of smooth and rough endoplasmic reticulum with disaggregation of polyribosomes and accumulation of mitochrondria and various dense bodies. SC was identified at 2 to 3 hours and was still recognizable at five days. When bilateral carotid artery occlusion lasted 5 to 6 minutes, SC was seen in the hippocampal Sommer sector and cerebral cortex, while ICC was restricted to the endfolium (h3-5). Unlike ICC, the frequency of SC was not related to the duration of ischemia but probably to the epileptic seizures (overt and subclinical) initiated by ischemia in the gerbil. These changes must be considered when the gerbil is employed as a model of experimental stroke.

Animals↗
PubMed1979

Experimental cerebral ischemia produces platelet aggregates.

Human studies indicate that transient platelet abnormalities accompany acute cerebral ischemia. Although these abnormalities may precipitate the ischemi process, ischemia could also alter platelet function. Platelets were therefore studied in gerbils subjected to 1 hour of unilateral carotid artery occlusion. Venous blood from five clinically affected gerbils contained more aggregated platelets (37.8% +/- 6.4) than did blood from eight unaffected animals (11.1% +/- 3.0; p less than 0.01). Platelets labeled with 3H-serotonin were increased in ischemic brain; the ratio of radioactivity in the ipsilateral versus contralateral hemisphere was greater in eight affected (1.09 +/- 0.03) than in 19 unaffected (1.00 +/- 0.01; p less than 0.02) animals. The radioactive serotonin was located predominantly within blood vessels. Cerebral ischemia thus stimulated the formation of platelet aggregates, a response which could contribute to the ischemic process.

Animals↗
PubMed1979

Delayed postischemic hypoperfusion: a potentially damaging consequence of stroke.

Cerebral blood flow (CBF) was assessed with radioactive butanol and antipyrine during and after 1 hour of unilateral carotid artery occlusion in gerbils. Animals with clinical evidence of stroke demonstrated a marked fall in ipsilateral CBF during occlusion "no-reflow" phenomenon did not develop; instead, blood flow returned to normal 5 minutes after the termination of carotid occlusion. Flow subsequently fell to half the control value, however, and remained depressed for several hours despite local metabolic demands. This delayed imbalance in energy supply and demand creates a potential for additional brain damage that might be prevented by appropriate therapeutic intervention.

Animals↗
PubMed1979

Outcome from severe neurological illness; should it influence medical decisions?

Most persons now accept the concept that when brain is dead, self is dead, and are willing to act accordingly. Much more difficult is to decide what to do when illness irreparably deprives the brain of cognitive functions. To facilitate less impassioned discussion of this question, 500 consecutive patients in coma from non-traumatic causes have been studied in the USA and Europe and the outcome has been compared against carefully selected early neurological signs. Medical coma was itself a dangerous sign, with only 15% of patients recovering independence within the first month. Within the first six hours, when most medical decisions are made about applying intensive care, neurological signs predicted with 95% confidence between the extremes of favourable and unfavourable outcomes in as many as a quarter of the patients. Only 5% of patients who failed to regain cognition by the end of one week regained any independence. Other more detailed guidelines were equally informative. If prognostic signs that confidently separate potentially favourable from unfavourable outcomes can be identified in severe neurological illness, perhaps society can more easily help medicine in reaching difficult ethical decisions.

Brain Diseases↗
PubMed1978

Reversed immunosorbents: a simple method for specific antibody immobilization.

A method is presented for permanently converting an antigen immunosorbent into a purified antibody immunosorbent which retains specific reactivity for antigen. Following immunological reaction of the antibodies in an antiserum with the antigen adsorbent, the specific immunoglobulin is chemically bound to the antigen by means of the divalent crosslinking agent. Various such agents, including glutaraldehyde, suberimidate, a diazide, and isocyanates, were employed. The conditions of preparation using the first two were optimized, and the characteristics of the resulting immobilized antibodies were investigated. Applications in radioimmunoassay, preparative chromatography, and affinity-constant studies discussed in view of the unique attributes of these reagents.

Animals↗