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Biomedical subjects

M Levine

Publications and source records attributed to M Levine.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.

Adult↗
PubMed1992

Individual dorsal morphogen binding sites mediate activation and repression in the Drosophila embryo.

The dorsal (dl) morphogen gradient is responsible for initiating the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the Drosophila embryo. dl encodes a sequence-specific DNA binding protein that belongs to the Rel family of transcription factors. Previous studies have shown that dl activates the mesoderm determinant twist (twi); here we use a combination of site-directed mutagenesis and P-transformation assays to demonstrate that it also functions as a direct transcriptional repressor of a second target gene, zerknüllt (zen). By exchanging dl binding sites between the promoters we show that activator sites from twi can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo. Evidence is also presented that the affinities of dl binding sites are important for the efficiency of repression, but are not the sole determinants of the threshold response to the dl gradient.

Animals↗
PubMed1992

Regulation of even-skipped stripe 2 in the Drosophila embryo.

In an effort to determine how crude gradients of transcriptional activators and repressors specify sharp stripes of gene expression in the early embryo, we have conducted a detailed study of even-skipped (eve) stripe 2. A combination of promoter fusions and P-transformation assays were used to show that a 480 bp region of the eve promoter is both necessary and sufficient to direct a stripe of LacZ expression within the limits of the endogenous eve stripe 2. The maternal morphogen bicoid (bcd) and the gap proteins hunchback (hb), Kruppel (Kr) and giant (gt) all bind with high affinity to closely linked sites within this small promoter element. Activation appears to depend on cooperative interactions among bcd and hb proteins, since disrupting single binding sites cause catastrophic reductions in expression. gt is directly involved in the formation of the anterior border, although additional repressors may participate in this process. Forming the posterior border of the stripe involves a delicate balance between limiting amounts of the bcd activator and the Kr repressor. We propose that the clustering of activator and repressor binding sites in the stripe 2 element is required to bring these weakly interacting regulatory factors into close apposition so that they can function both cooperatively and synergistically to control transcription.

Animals↗
PubMed1992

Hemostatic alterations in cancer patients.

Nearly all patients with cancer manifest laboratory evidence of hypercoagulability and some develop clinical thromboembolic disease (TED). Routine laboratory studies of blood coagulation have been performed in several large, prospective trials of the use of anticoagulant drugs in cancer treatment. The results of these studies, as well as data from several smaller studies of more sensitive tests of hypercoagulability [e.g. fibrinopeptide A (FPA); thrombin-antithrombin (TAT) complexes; prothrombin fragment F1 + 2)], indicate that the levels of some clotting proteins parallel disease activity. However, no studies of sound methodologic design have yet been performed to indicate that any of these tests of blood coagulation can serve as adequate predictors of TED in patients with cancer. In addition to the important role played by tumor-related procoagulants, several other mechanisms may be involved in the pathogenesis of thromboembolic events in patients with cancer, including stasis and endothelial damage. Considerable variability in the relative importance of these mechanisms in the pathogenesis of TED may exist among patients with different types of cancer. The risk for TED associated with surgical procedures in cancer patients is substantial and prophylactic antithrombotic therapy should be considered for most of these patients. Chemotherapy and hormonal therapy of cancer probably increases the likelihood of TED, particularly in those subjects with indwelling venous catheters. This risk has been particularly well-studied in patients with breast cancer treated with tamoxifen plus cytotoxic drugs. The pathogenic mechanisms may be complex but vascular injury is likely as a proximate cause of venous access catheter thrombosis and can be prevented with low dose coumadin therapy. The utility of low dose coumadin anticoagulation in reducing the risk for TED during breast cancer treatment is unknown but is currently being tested in a large, multiinstitutional study. Since chronic coumadin anticoagulation of cancer patients, and single pulse dose heparin prior to intravenous chemotherapy, both prevent thrombin generation, these agents may be of use in reducing the risk of chemotherapy-associated thrombosis. Prophylactic anticoagulation should be considered for high risk patients.

Antineoplastic Agents↗
PubMed1992

Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine.

The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aroC strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.

Administration, Oral↗
PubMed1992

Ectopic expression of the Drosophila tramtrack gene results in multiple embryonic defects, including repression of even-skipped and fushi tarazu.

The tramtrack (ttk) gene of Drosophila encodes 69-kDa and 88-kDa proteins through alternative splicing of the primary ttk transcript. The two proteins share a common amino-terminal sequence, but contain different carboxy-terminal portions, each of which has a distinct zinc finger domain with a unique DNA binding specificity. The 69-kDa ttk protein has been shown to bind multiple sites within important regulatory elements of the pair-rule genes even-skipped (eve) and fushi tarazu (ftz), and it has been suggested that this protein may function as a repressor of ftz transcription. Here we present evidence that the 69-kDa ttk protein can indeed repress expression not only of ftz, but also of eve. Ectopic expression of the 69-kDa protein, but not of the 88-kDa form, was found to nearly abolish the striped patterns of expression of both eve and ftz in transgenic embryos. These findings, coupled with our detection of significant levels of ttk protein in ovaries and 0-2-h embryos, support the idea that maternally supplied ttk protein serves to prevent premature activation of eve and ftz, thereby helping to establish the timing of the onset of zygotic expression of these two genes. Furthermore, gross defects in the larval cuticle resulting from misexpression of the 69-kDa protein suggest that this protein performs additional functions in the early embryo.

Animals↗
PubMed1992

A parkinsonian syndrome induced in the goldfish by the neurotoxin MPTP.

Parkinson's disease has been modeled in humans, lower primates, and to a lesser extent in some other vertebrates by administration of the potent neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine). The MPTP model has thus drawn considerable attention as a system to search for anti-Parkinson's disease drugs, although the cost and scarcity of primates has limited extensive applications. We now report that a parkinsonian syndrome can be elicited in the common goldfish (Carassius auratus) by a single dose of MPTP. The syndrome is characterized by profound bradykinesia (slow movement), the full extent of which is reached 3 days after MPTP administration. The reduction in movement is paralleled by loss of dopamine and norepinephrine from the forebrain and midbrain and in other brain regions as well. The toxic oxidative product of MPTP, MPP+, is also accumulated predominantly in forebrain and midbrain, and pretreatment with the monoamine oxidase blocker tranylcypromine substantially reduces accumulation of the toxic metabolite. A barely perceptible coarseness in balance adjustment also occurs in treated animals. The MPTP-treated goldfish recover normal movement and normal brain monoamine levels within 10-13 days after administration of the drug. We interpret these and other data to indicate that MPTP can induce a Parkinson's disease-like syndrome in the goldfish that is similar in many aspects to the syndrome induced by MPTP in humans and other primates. This remarkable parallel may permit the goldfish to supplement expensive and scarce primates for the purpose of searching and screening neuroprotective drugs with specific relevance to Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo.

rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. It is initially expressed before the completion of cellularization in lateral stripes within the presumptive neuroectoderm. Here, we present evidence that the maternal morphogen dorsal (dl) acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including twist (twi), to activate rho in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by snail (sna), which is also a direct target of the dl morphogen. A 300-bp region of the rho promoter (the NEE), which is sufficient for neuroectoderm expression, contains a cluster of dl and b-HLH activator sites that are closely linked to sna repressor sites. Mutations in these binding sites cause genetically predicted changes in the levels and limits of rho expression. In particular, the disruption of sna-binding sites causes a derepression of the pattern throughout ventral regions, providing evidence that sna is directly responsible for establishing the mesoderm/neuroectoderm boundary before gastrulation. The tight linkage of activator and repressor sites in the rho NEE is similar to the arrangement of binding sites observed in the even-skipped stripe 2 element, which is regulated by bicoid (bcd). This suggests that the dl and bcd morphogens use a similar mechanism to make stripes in the Drosophila embryo.

Animals↗
PubMed1992

Prophylactically equivalent doses of Enoxaparin and unfractionated heparin inhibit in vivo coagulation to the same extent.

This study compared how Enoxaparin and unfractionated (UF) heparin influenced in vivo coagulation in patients randomized to receive, by twice daily subcutaneous injections, either 30 mg of Enoxaparin or 7500 I.U. of UF heparin after elective hip surgery. These two regimens were equally effective in reducing the incidence of post-operative deep vein thrombosis DVT. We compared the concentrations of endogenous thrombin-antithrombin III in pre- and post-surgical plasmas to determine how each prophylactic regimen influenced prothrombinase activity in vivo, and found the same concentrations of endogenous thrombin-antithrombin III in post-heparin and post-Enoxaparin plasmas. However, significantly higher concentrations of endogenous thrombin-antithrombin III were found in pre- and post-surgical plasmas of patients who developed post-operative DVT than the levels found in comparable plasmas of patients who remained DVT-negative, regardless of the drug received for prophylaxis. Human factor Xa was added to an equal volume of each patient's plasmas and the amount of added enzyme inactivated by antithrombin III measured using an enzyme-linked immunosorbent assay for factor Xa-antithrombin III. Post-heparin and post-Enoxaparin plasmas inactivated approximately 4 times more factor Xa than the pre-surgical plasmas, regardless of the clinical outcome. Thus, before and after surgery, a higher than normal in vivo prothrombinase activity may be a significant risk factor for developing post-operative DVT.

Antithrombin III↗
PubMed1992

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.

Amino Acid Sequence↗
PubMed1992

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.

Animals↗
PubMed1992

The relationship between environmental temperature, metabolic rate, sleep state, and evaporative water loss in infants from birth to three months.

We have investigated the effect of changing environmental temperature on metabolic rate, sleep state, and water loss in a longitudinal study of 22 lightly clothed babies from 2 d to 3 mo of age. Studies were performed in a modified barometric plethysmograph while recording sleep state, oxygen consumption, and skin and axillary temperatures. Oxygen consumption was higher in rapid eye movement sleep than in quiet sleep at all ages and varied widely between infants at each temperature. Within the first week, there was a 19% rise in oxygen consumption on cooling to 19-22 degrees C during rapid eye movement sleep and a 6% rise during quiet sleep. The median duration of quiet sleep periods was reduced from 17 to 12 min on cooling within the first week. No such change was seen at 1, 2, and 3 mo. Axillary temperature was reduced at 3 mo during cooling. This may be a part of normal patterns of change in temperature during sleep, unrelated to cooling. At each age, total evaporative water loss fell linearly with falling environmental temperature both within and below the temperature range at which metabolic rate was minimal. The evaporative water losses were greater than expected and suggested that sweating was occurring, both at temperatures at which metabolic rate was minimal and at those at which it was increased. The metabolic response to cooling and the process of sweating appear to be in dynamic equilibrium across this temperature range. Thus, it was not possible to define a temperature range over which both metabolic rate and evaporative water loss were at minimum values.

Body Temperature Regulation↗
PubMed1992

The bicoid and dorsal morphogens use a similar strategy to make stripes in the Drosophila embryo.

The anterior-posterior (A-P) and dorsal-ventral (D-V) axes of the early Drosophila embryo are established by two key maternal morphogens: bicoid (bcd) and dorsal (dl), respectively. The bcd protein is expressed in a broad concentration gradient along the A-P axis, with peak levels present at the anterior pole, while dl is expressed in a gradient along the D-V axis with peak levels along the ventral surface. The two morphogens are unrelated and their gradients are formed by distinct processes. Nonetheless, we have obtained evidence that they generate sharp on/off stripes of target gene expression through a similar mechanism. Both morphogens establish overlapping patterns of transcriptional activators and repressors in the early embryo. The activators and repressors bind to closely linked sites within short (300 to 500 bp) target promoter elements that have the properties of on/off switches. The activators act in concert with the morphogen to define a broad region where target genes can be initiated. Borders of target gene expression are established by the repressors, resulting in the formation of stripes.

Animals↗
PubMed1992

Opioid receptor modulation of postexercise hypotension.

Previous studies of both hypertensive and normotensive individuals have indicated a prolonged reduction in blood pressure for several hours after aerobic exercise. In related studies of spontaneously hypertensive rats, this postexercise hypotension has been prevented with naloxone. The purpose of the present investigation was to examined whether the postexercise hypotension may be reversed by antagonism of opioid sensitive receptors with naloxone in normotensive humans. Eight males 22-34 yr of age, participated in two 60-min cycling trials at 60% of VO2 peak, followed by 29 min of recovery. Beginning at 7-min recovery, naloxone or saline (control) was administered intravenously through an indwelling catheter. Blood pressure and heart rate were monitored every 15 min during exercise and every 2 min during recovery. Heart rate was significantly elevated (P < 0.05) over basal levels for the first 11 min of recovery, but from 13 to 29 min was not different from that measured at rest. In both trials, after 11 min of recovery, systolic and mean arterial blood pressures were significantly (P < 0.05) lower than pre-exercise levels (9 +/- 1 mm Hg and 4 +/- 1 mm Hg, respectively). Injection of naloxone (0.1 mg.kg-1) reversed the hypotensive response. However, the reversal was transient, lasting from minutes 15 to 27. Since naloxone reverses postexercise hypotension, opioid sensitive receptors appear to be involved in the reduction in systolic blood pressure following a single bout of submaximal exercise in normotensive humans.

Adult↗