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M Levine

Publications and source records attributed to M Levine.

At least 181 records · Page 10Linked to original sources
PubMed1996

Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin.

Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC. The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent. The reducing activity was purified to homogeneity as an 11 kDa protein. The protein had a specific activity of 3 mumol/min per mg of protein and was glutathione dependent. Kinetic experiments showed that the protein had a K(m) for glutathione of 2.0 mM and a K(m) for dehydroascorbic acid of 250 microM. Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5. Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin. By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library. Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme. Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates. From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.

Amino Acid Sequence↗
PubMed1996

Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.

Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value.

Adult↗
PubMed1996

Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila.

The early Drosophila embryo provides a unique system for the analysis of transcriptional repression since a broad spectrum of repressors are distributed in spatially distinct patterns. Krüppel (Kr) and snail (sna), two zinc finger repressors, are essential for segmentation and for the establishment of the mesoderm/neuroectoderm boundary, respectively. Both repressors were examined in the context of synthetic gene complexes containing modular promoters and divergently transcribed reporter genes. These studies indicate that Kr and sna function as short-range repressors, which can mediate either quenching or direct repression of the transcription complex, depending on the location of repressor sites. When located within an upstream enhancer, the repressor locally quenches nearby activators and permits other enhancers to interact with the transcription complex (enhancer autonomy). In contrast, when bound to promoter-proximal regions the repressor functions in a dominant fashion and blocks multiple enhancers. Local quenching and dominant repression require close linkage (<100 bp) of the repressor with either upstream activators or the transcription complex. These studies establish short-range repression as a flexible form of gene regulation and suggest that the key distinction among repressors is their range of action.

Animals↗
PubMed1996

A comparison of low-molecular-weight heparin administered primarily at home with unfractionated heparin administered in the hospital for proximal deep-vein thrombosis.

BACKGROUND: Patients with acute proximal deep-vein thrombosis are usually treated first in the hospital with intravenous standard (unfractionated) heparin. However, the longer plasma half-life, better bioavailability after subcutaneous administration, and more predictable anticoagulant response of low-molecular-weight heparins make them attractive for possible home use. We compared these two approaches. METHODS: Patients with acute proximal deep-vein thrombosis were randomly assigned to receive either intravenous standard heparin in the hospital (253 patients) or low-molecular-weight heparin (1 mg of enoxaparin per kilogram of body weight subcutaneously twice daily) administered primarily at home (247 patients). The study design allowed outpatients taking low-molecular-weight heparin to go home immediately and hospitalized patients taking low-molecular-weight heparin to be discharged early. All the patients received warfarin starting on the second day. RESULTS: Thirteen of the 247 patients receiving low-molecular-weight heparin (5.3 percent) had recurrent thromboembolism, as compared with 17 of the 253 patients receiving standard heparin (6.7 percent; P=0.57; absolute difference, 1.4 percentage points; 95 percent confidence interval, -3.0 to 5.7). Five patients receiving low-molecular-weight heparin had major bleeding, as compared with three patients receiving standard heparin. After randomization, the patients who received low-molecular-weight heparin spent a mean of 1.1 days in the hospital, as compared with 6.5 days for the standard-heparin group; 120 patients in the low-molecular-weight- heparin group did not need to be hospitalized at all. CONCLUSIONS: Low-molecular-weight heparin can be used safely and effectively to treat patients with proximal deep-vein thrombosis at home.

Aged↗
PubMed1996

Child age and caseworker attention in child protective services investigations.

Infants and toddlers are at increased risk for severe/fatal abuse, often at the hands of male perpetrators. This paper examined whether child maltreatment cases involving younger children receive more casework services, overall caseworker activity (e.g., home visits, phone contacts), and are more likely to be substantiated, particularly if a male perpetrator is involved. The randomly selected sample consisted of 293 child abuse and neglect reports in a large county in Western New York in 1993. Hierarchical regression analyses showed that younger children generally receive more overall services and caseworker activity, and that while age makes a significant contribution after the decision has been made to substantiate a case, it does not have the same effect on the initial decision of whether or not to substantiate. Gender of the perpetrator did not reliably predict caseworker attention.

Adolescent↗
PubMed1996

Characterization of an immunodeficiency mutant in Drosophila.

Drosophila immunity and embryogenesis appear to be linked by an evolutionarily ancient signalling pathway, which includes the Rel-domain transcription factors Dif and dorsal, respectively, as well as a common inhibitor, cactus. Previous genetic screens have centered on maternal mutants that disrupt the dorsal pathway. In an effort to identify additional components that influence Rel-domain gene function we have conducted a search for immunodeficiency mutants in Drosophila. One such mutant, which maps near the Black cells (Bc) gene, causes a severe impairment of the normal immune response, including attenuated induction of several immunity genes. Survival assays indicate a positive correlation between the induction of these genes, particularly diptericin, and resistance to bacterial infection. These studies are consistent with the notion that insect anti-microbial peptides work synergistically by binding distinct targets within infecting pathogens. Evidence is also presented that non-specific acquired immunity results from the persistence of bacterial metabolites long after primary infection. We discuss the potential usefulness of this study with regard to the identification of conserved components of Rel signalling pathways.

Animals↗
PubMed1996

Transcriptional repression in development.

It is becoming increasingly clear that transcriptional repression is at least as important as transcriptional activation for establishing cell-type specific patterns of gene expression during embryogenesis. Recent studies in Drosophila suggest that repressors fall into two categories, short-range and long-range repressors. The former permit enhancer autonomy in modular promoters, whereas the latter function in a dominant fashion to silence multiple enhancers.

Animals↗
PubMed1996

Threshold responses to the dorsal regulatory gradient and the subdivision of primary tissue territories in the Drosophila embryo.

Dorsoventral patterning in Drosophila is initiated by the maternal regulatory factor dorsal (dl), which is a member of the Rel family of transcription factors. dl functions as a transcriptional activator and repressor to establish different territories of gene expression in the precellular embryo. Differential regulation of dl target genes may be essential for subdividing each tissue territory (the presumptive mesoderm, neuroectoderm, and dorsal ectoderm) into multiple cell types in older embryos. Different patterns of snail (sna) and decapentaplegic (dpp) expression help define the limits of inductive interactions between the mesoderm and dorsal ectoderm after gastrulation. Similarly, the differential regulation of short gastrulation (sog) and dpp may be decisive in the initial subdivision of the dorsal ectoderm, whereas different limits of gene expression within the neuroectoderm might provide the basis for the subsequent subdivision of this tissue into ventral and lateral regions.

Animals↗
PubMed1996

Midazolam following open heart surgery in children: haemodynamic effects of a loading dose.

Our objective was to establish the safety and effectiveness of a loading dose of midazolam for postoperative sedation of children recovering from open heart surgery; a prospective randomized placebo-controlled double-blind study was done with subjects randomized to three groups according to loading dose. I = 0.08 mg.kg-1; II = 0.04 mg.kg-1; and III = 0.00 mg.kg-1 (placebo). An open label continuous midazolam infusion protocol followed. Haemodynamic parameters were monitored. The study was discontinued following an adverse event involving the 23rd subject. When data for all 23 subjects were combined, there was a mean decrease of 10% in blood pressure (BP) 30 min after the loading dose (P < 0.001). Heart rate change was less significant. Clinicians identified four hypotensive episodes as temporally associated with the midazolam load, two each in Groups I (0.08 mg.kg-1) and III (placebo). One subject in Group I (the 23rd) became hypotensive within five min of receiving the loading dose, had a difficult clinical course and died four weeks postoperatively. We cannot conclude that the loading dose of midazolam had any systematic haemodynamic effect in our study population. Although the clinical course of the 23rd subject suggests a subset of more susceptible children (those who receive opioid analgesia with midazolam, are volume-restricted, and/or undergo more complex forms of surgical correction), many critical care patients are inherently physiologically unstable, and concluding clinically that blood pressure fluctuation is drug related may be erroneous.

Blood Pressure↗
PubMed1996

The evaluation of various mathematical RBC indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis.

The differentiation between thalassemic and non-thalassemic microcytosis has important clinical implications in hematology and medicine. A simplified index, based on red cell parameters derived from automated blood cell analyzers, which could be used to discriminate between microcytic patients with a high probability of thalassemia minor and those with a low probability, would be an extremely useful tool. Five mathematical indices have been proposed as useful for this purpose. These are the: Bessman index, Shine and Lal index, England index, Mentzler index, and mean cell volume (MCV) alone. This study was designed to prospectively evaluate the efficacy of these indices. Patient samples were chosen every fourth day from all patient samples referred to the hematology laboratory at St. Joseph's Hospital over a 6-month period. All patient samples with an MCV < 80 fL and age > or = 18 years were considered eligible for the study. After enrollment and laboratory analysis were complete sensitivities and specificities were calculated for each of the indices using a variety of cut-off values and receiver operator characteristic (ROC) curves were constructed. Based on statistical analysis of the area under these curves, the authors conclude that MCV alone is as effective as the Mentzler and Shine and Lal indices in selecting microcytic patient samples with a high probability of thalassemia minor for thalassemia testing. They also conclude that the Bessman index and the England index are ineffective indices for this purpose.

Adult↗
PubMed1996

An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies.

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.

Animals↗
PubMed1996

Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia by in situ reverse transcriptase PCR.

One of the defining characteristics of herpes simplex virus type 1 (HSV-1) infection is the ability of the virus to establish a lifelong latent state in neurons. We previously demonstrated (R. Ramakrishnan, A.J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994) by in situ PCR that many more neurons contain viral genomes than are detected by in situ hybridization for HSV latency-associated transcripts (LATs). To determine whether all cells which contain genomes express LATs, we examined trigeminal ganglia for LATs 1 and 8 weeks after corneal scarification with ribonucleotide reductase-deficient HSV-1 by in situ reverse transcriptase PCR. The number of LAT-positive cells detected by in situ reverse transcriptase was substantially greater than the number of cells positive by in situ hybridization and appeared to be similar to the number of cells containing HSV genomes by in situ PCR and the number of ganglionic neurons that project to the cornea as detected by retrograde labeling with Fluorogold. These results demonstrate LAT expression in many neurons containing HSV-1 genomes.

Animals↗
PubMed1996

The eve stripe 2 enhancer employs multiple modes of transcriptional synergy.

Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo. The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo, bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively. Here, we report the findings of extensive cis- and trans- complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos. This study reaches two primary conclusions. First, the strip 2 enhancer is inherently 'sensitized' for repression by gt. We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex. Second, we find that the synergistic activity of hb and bcd is 'promiscuous'. For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer. This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.

Animals↗
PubMed1996

Inhibitory action of palytoxin on ascorbic acid transport into cultured bovine adrenal chromaffin cells.

The effect of palytoxin on the transport of ascorbic acid into cultured bovine adrenal chromaffin cells was examined by measuring the accumulation of radiolabeled ascorbic acid within cells. Ascorbic acid transport into these cells was inhibited by palytoxin in a concentration-dependent manner, and this inhibitory action of palytoxin was shown to be noncompetitive and irreversible. Neither the Na+/K+-pump activity in the intact cells nor the Na+,K+-adenosine 5'-triphosphatase activity in the plasma membranes was significantly influenced by this toxin at concentrations inhibiting ascorbic acid transport. In contrast to the effect of palytoxin on ascorbic acid transport, glucose transport into these cells was not significantly affected by this toxin. These findings indicate that palytoxin can inhibit ascorbic acid transport into adrenal chromaffin cells without affecting Na+,K+-adenosine 5'-triphosphatase activity in the plasma membranes. Furthermore, because palytoxin discriminated between ascorbic acid transport and glucose transport, the data provide new evidence that the transport of ascorbic acid and that of glucose may be mediated by different mechanisms in the adrenal medullary cell.

Acrylamides↗
PubMed1996

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency.

The nuclear matrix plays a critical role in DNA replication, gene transcription and RNA processing. Transcriptionally active genes are usually associated with the nuclear matrix through DNA sequences, matrix attachment regions or MARs, which tether looped DNA to the matrix. In stable transfection and in transgenic mice MAR elements placed at the flanks of genic constructs may enhance expression and insulate against position effect variability, suggesting that independent units of transcription are established insulated from the regulatory controls of their neighbors. Herpes simplex virus type 1 (HSV-1) establishes lifelong latency in the infected host. Latency repression of viral genes extends to foreign genes incorporated into the viral genome. We report here a test of the hypothesis that MAR elements, flanking a foreign gene in the HSV-1 genome, would act to insulate it from latency repression, achieving long-term expression. A recombinant virus was produced which has an expression construct inserted into the HSV-1 genome at the Us3 locus. The expression construct consists of the A MAR element on one flank, an HIV-LRT driving the lacZ gene and the B MAR element on the other flank. The A MAR element is a 3 kb pair fragment of the 5' portion of the chicken lysozyme gene and the B MAR element is a 2.6 kb pair fragment from the 5' end of the human beta-globin gene locus control region. The LTR is derived from a human immunodeficiency virus isolated from the brain of an AIDS patient. Virus was stereotactically injected in the hippocampus, olfactory bulb and striatum of rat brains. Intense blue reaction product indicating beta-galactosidase activity was found in cells in each injected area at 2 days after injection. At 14 days after injection beta-galactosidase activity was no longer detected at any of the injected sites. We conclude that the MAR element construct did not escape latency repression.

Animals↗