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Biomedical subjects

N S Levy

Publications and source records attributed to N S Levy.

At least 19 recordsLinked to original sources
PubMed1995

Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor and endothelial cell-specific mitogen, is up-regulated by hypoxia. However, the mechanism(s) responsible for hypoxic induction of VEGF has not been clearly delineated. We report that the steady state VEGF mRNA levels are increased 12 +/- 0.6-fold, but the transcriptional rate for VEGF is increased only 3.1 +/- 0.6-fold by hypoxia in PC12 cells. In order to investigate cis-regulatory sequences which mediate this response to hypoxia, we cloned the rat genomic sequences encoding VEGF and identified a 28-base pair element in the 5' promoter that mediates hypoxia-inducible transcription in transient expression assays. This element has sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin 3' enhancer. Post-transcriptional mechanisms have also been suggested to play a role in the hypoxic induction of VEGF. Evidence is provided that a frequently used polyadenylation site is 1.9 kilobases downstream from the translation termination codon for rat VEGF. This site is 1.5 kilobases further downstream from the polyadenylation site previously reported for VEGF. This new finding reveals sequence motifs in the 3'-untranslated region that may mediate VEGF mRNA stability.

Animals↗
PubMed1995

Regulation of vascular endothelial growth factor in cardiac myocytes.

Collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease, thereby protecting the myocardium from ischemia. Collateral vessel formation is the result of angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic factor. In the present study, VPF/VEGF mRNA and protein were demonstrated to be markedly stimulated in primary rat cardiac myocytes in vitro in response to reduction of the oxygen tension to 1% or inhibition of the electron transport chain. Four isoforms of VPF/VEGF were coordinately regulated by hypoxia, including a novel isoform not previously described. Phorbol ester and the depolarizing agent veratridine, stimulators of protein kinase C and calcium influx, respectively, were found to markedly increase VPF/VEGF mRNA expression in cardiac myocytes. Forskolin, a potent stimulator of adenylate cyclase, produced a small but significant increase in VPF/VEGF mRNA expression in the cardiac myocytes. However, only H7, an inhibitor of protein kinase C, inhibited the hypoxic induction of VPF/VEGF mRNA; inhibitors of calcium influx and the calcium-calmodulin-dependent protein kinase II as well as inhibition of protein kinase A did not block the hypoxic induction of VPF/VEGF mRNA. This suggests that more than one signal transduction pathway is involved in regulating VPF/VEGF expression. The sensor that regulates the expression of hypoxia-responsive genes has been proposed to be a heme protein. Consistent with this model, transition metals initiate a genetic program similar to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The molecular basis of signal transduction in olfactory sensory neurons.

Contributions from a wide spectrum of experimental systems have resulted in a dramatic increase in our understanding of this old and most enigmatic of the sensory systems. Many of the components of the odorant-induced transduction cascade have now been cloned, and the biochemistry, pharmacology, and regulatory mechanisms are being addressed in a logical fashion. One of the first priorities is to establish that the large family of putative receptor proteins described by Buck and Axel (1991) do, in fact, bind odorants. The ability to express members of this receptor family at high levels in the mammalian expression system is a first step in this direction. Determining specific ligand-receptor relationships is an extremely challenging task given the diversity of odorants able to be perceived and the potentially large size of the family of receptors. The role of other proteins in odorant presentation and processing, such as odorant binding protein produced in the lateral nasal gland (Pevsner et al., 1988), can be explored. A fascinating issue to be resolved is that of the distribution of receptor molecules within the population of olfactory sensory neurons. Does one cell express only one receptor, a small repertoire of receptors, or indeed the entire family? These questions can now be answered using a combined approach with in situ hybridization, immunocytochemistry, and single cell PCR techniques. One model of receptor distribution would provide for discrimination of a particular odorant by higher order analysis of the pattern of receptor neuron firing within the neuroepithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.

Somatic mutation in immunoglobulin genes is localized to a 2-kilobase region of DNA surrounding and including rearranged variable (V), diversity, and joining (J) gene segments encoding heavy and light chains. To examine the structural basis for targeted mutation, we developed an assay to score mutation on plasmid substrates by using a reporter gene: a bacterial gene encoding an amber-suppressor tRNA molecule was placed 3' of a rearranged kappa VJ gene within the boundaries of mutation. The reporter gene is exquisitely suited for mutational analysis because it is only 200 base pairs (bp), which should not greatly disrupt structure of the immunoglobulin locus, and gene function depends on secondary structure, which means mutation can be scored in many different nucleotide positions. The plasmid was used to make transgenic mice, which were then immunized. The shuttle vector was retrieved by plasmid rescue into an indicator strain of Escherichia coli that contained an amber mutation in its beta-galactosidase gene. Integrity of the tRNA molecule was monitored by colony color, which permitted many transformants to be screened visually. Mutations were not seen in DNA from a transfected B-cell line grown in vitro or in DNA from nonlymphoid tissue of transgenic mice, indicating that the reporter gene was stable during cell division and DNA manipulations. However, when the transgenic mice were immunized, DNA from splenic B cells contained point mutations in the reporter gene at a frequency of 10(-3) per transformant. Sequence analysis of 17 mutated transgenes revealed that the mutations were 1- and 2-bp deletions in the tRNA gene, and one plasmid had an additional 2-bp deletion in the V gene. In contrast, previous studies have shown that mutations in endogenous VJ genes are predominantly nucleotide substitutions and have only 6% deletions. Two other plasmid constructs were analyzed in transgenic lines: no mutations were found when the tRNA gene was placed distal to the VJ gene, and no mutations were seen when the immunoglobulin promoter was deleted. Although we lack direct evidence that the deletions in the tRNA gene are caused by the same mechanism that acts on VJ genes, we have shown that mutations in this assay occur in a manner consistent with immunoglobulin-specific mutation in that they are found in splenic B cells and not in tail tissue, depend on position next to the VJ gene, and require transcription of the VJ gene.

Animals↗
PubMed1991

Signal transduction in olfactory neurons.

Recent efforts in our laboratory have focused on cloning the molecular components involved in the cAMP-mediated pathway of olfactory signal transduction. These efforts have resulted in the isolation of olfactory-specific forms of a G protein, an adenylyl cyclase, and a cyclic nucleotide-gated cation channel. Functional expression of each of these proteins in vitro confirms their ability to carry out the function ascribed to them as part of a second-messenger cascade. Putative odorant-receptor molecules which constitute the first step in odorant signal transduction have now been cloned. We have generated oligonucleotide probes which recognize a population of olfactory receptors apparently more heterogeneous than those previously reported. These probes should enable us to answer questions regarding the number of different receptors expressed per cell as well as the nature of receptor-ligand specificity.

Adenylyl Cyclases↗
PubMed1989

Early onset of somatic mutation in immunoglobulin VH genes during the primary immune response.

The dynamics of somatic mutation in Ig variable genes was investigated in order to define a population of B cells undergoing mutation. BALB/cJ mice were injected with PC-KLH, and splenic RNA was prepared 5, 7, and 13 d later. The mRNA was annealed to gamma constant region primers to make cDNA transcripts encoding VH genes. 103 cDNA clones corresponding to 18 different genes from the VH7183, VH3660, and VHS107 subfamilies were sequenced to identify mutation. VH genes had a low level of mutation on day 5 after immunization and accumulated more mutation by day 7 at a rate of 10(-3) mutations per nucleotide per generation. However, by day 13, the number of mutations per gene did not increase, and most of the substitutions encoded replacement amino acid changes that were clustered in the hypervariable regions, indicating that the mutational process was less active during the second week and that antigen selection had occurred. The data are consistent with a developmentally regulated mechanism in which mutation is activated during the first week of the primary immune response for a limited time period, after which selection acts to preserve the beneficial mutants.

Animals↗
PubMed1989

Transscleral YAG cyclocoagulation of the ciliary body for persistently high intraocular pressure following penetrating keratoplasty.

Nine postkeratoplasty eyes with persistent intraocular pressure (IOP) elevation were treated under an investigational protocol by transscleral YAG cyclocoagulation (TSYCC). All eyes were using the maximally tolerated antiglaucomatous medical therapy. Some eyes also had prior antiglaucomatous surgery, including cyclocryotherapy. All of these eyes were considered poor candidates for filtration. Laser applications were applied in a single session at 32 to 36 equally spaced locations throughout 360 degrees, 2.5 mm posterior to the limbus. The energy employed was between 7 and 10 joules at each site. Total energy varied between 256 and 352 joules per eye. The results and complications associated with this procedure are described in nine eyes with a minimum follow-up of 3 months and a median follow-up of over 6 months. Pressure control was achieved in all eyes. At the energy levels employed, hypotony (IOP less than 3 mm Hg) developed in two of the nine eyes, both of which had prior cyclocryotherapy. IOP was between 5 and 19 mm Hg in the remaining eyes. One of these required further medication (betaxolol) to obtain IOP less than 20 mm Hg, and one other developed a late pressure elevation due to pupillary block. Although these results confirm the efficacy of TSYCC, our outcomes suggest that total energy should be limited to less than 256 joules at the time of initial treatment in order to reduce the incidence of hypotony. Eyes with prior cyclodestructive therapy appear most likely to develop hypotony and should, accordingly, be treated with reduced total energy. Patent iridotomies are required in pseudophakic eyes, as pupillary block can develop due to fibrinous iridocyclitis.

Aged↗
PubMed1987

Impaired immunity in vitamin A-deficient mice.

Vitamin A deficiency was produced in mice and was used to investigate the role of vitamin A in immune function. Cellular immunity, as measured by delayed-type hypersensitivity, diminished in early deficiency before weight and appetite changes occurred and declined further as the deficiency progressed. Humoral immunity, as measured by serum immunoglobulin M (IgM) responses to a protein antigen (hemocyanin), also declined. The kinetics of antibody production were unaffected by the deficiency. The T-cell number remained unchanged, but B-cell and macrophage numbers were increased in vitamin A--deficient mice. Surface expression of membrane glycoproteins (Thy-1, Lyt-1, Lyt-2, L3T4, IgM, Mac-1) was unchanged by the deficiency, as were lymphocyte numbers and distribution. The results suggest that vitamin A deficiency is associated with a functional immune system defect.

Animals↗
PubMed1987

Somatic mutation in anti-phosphorylcholine antibodies.

A detailed analysis of the genes and proteins that participate in the murine immune response to PC has provided key insights at the structural level into the phenomenon of somatic mutation in B cells. Most anti-PC antibodies are encoded by 1 VH gene of the S107 subfamily, and 3 VK genes, VKT15 of the VK22 subfamily, VKM3 from the VK8 subfamily, and VK167 from the VK24 subfamily. No mutation was detected in these genes until the 2nd wk after immunization, indicating that mutation is under developmental control. The protein sequences of 73 heavy and light chains derived from the secondary response support the concept of developmental activation of mutation after antigen stimulation. No mutation was found in the IgM antibodies, whereas half of the IgG and IgA antibodies had mutation. Most of the mutated antibodies had higher affinity for antigen than their germline counterparts, which suggests that the major role of somatic mutation is to increase affinity rather than to create new specificities. Nucleotide sequencing established two hallmarks of mutation in immunoglobulin genes: mutations are targeted to a 1 kilobase region surrounding and including the rearranged variable gene, and they occur at an extraordinary frequency of 10(-2) nucleotide substitutions. Mutation is probably caused by DNA repair, and may occur during error-prone repair of nicked DNA around the variable gene or during mismatch repair of misaligned structural intermediates. The elucidation of this remarkable mechanism clearly requires studies of a more dynamic character. Two major questions that need to be answered are: what targets mutation to the variable gene, and what enzymes are involved?

Animals↗
PubMed1985

Matched comparison of Goldmann perimetry and automated two-zone suprathreshold Dicon perimetry in open-angle glaucoma.

Goldmann kinetic perimetry is a commonly used measurement of visual field defects in open-angle glaucoma. The many new automated perimeters should be assessed against this common standard with a view to comparing their sensitivity in making clinical judgments of the presence and extent of a visual field defect. This study compares the Coopervision Dicon 2000 perimeter and its automated two-zone suprathreshold program with Goldmann kinetic perimetry in 26 patients with open-angle glaucoma. The two-zone suprathreshold testing program on the Coopervision Dicon perimeter was found to be at least as sensitive as the Goldmann kinetic perimeter in identifying the presence and extent of visual field defects in open-angle glaucoma.

Glaucoma, Open-Angle↗
PubMed1984

Displacement of optic nerve head in response to short-term intraocular pressure elevation in human eyes.

Mechanical movement, reflecting compression within the lamina cribrosa, has been suggested as an initiating event in the production of damage in glaucoma. Reversible movements within the lamina cribrosa were sought and studied by characterizing the displacement of fine platinum wire following short-term elevation of intraocular pressure in 13 enucleated human eyes. With IOP elevation, maximum movement in the optic nerve occurred near its center, with minimum movement at its periphery. The difference in movement between these two locations increased with increases in IOP. However, the greatest displacement per unit of IOP elevation occurred at the lowest IOP. Resistance to further displacement occurred as higher IOP was reached. The differential mechanical displacement observed may initiate damage in open-angle glaucoma by causing compression of the vessels and/or shearing of axons within the lamina cribrosa.

Adult↗
PubMed1981

Displacement of the optic nerve head. Response to acute intraocular pressure elevation in primate eyes.

Mechanical compression of axons within the lamina scleralis has been suggested as a mechanism of damage in glaucoma. Movement within the optic nerve head was studied after acute intraocular pressure elevation in the enucleated primate eye. Fine platinum wire was positioned with the lamina scleralis and displacement characterized after IOP elevation. These studies demonstrate the following: (1) retrodisplacement increases significantly with increasing pressure, (2) maximum retrodisplacement occurs at the center and minimum retrodisplacement occurs at the periphery of the optic nerve, (3) retrodisplacement at the position of minimum movement in the optic nerve is indistinguishable from that in the sclera, (4) 67% of the net retrodisplacement occurs after a 15-mm Hg increase in IOP, and (5) tangential displacements within the lamina scleralis also increase with increasing pressure but are only 50% of the magnitude of retrodisplacements.

Animals↗
PubMed1979

Non-diabetic retinal abnormalities in chronic pancreatitis.

To determine whether retinal abnormalities occur in patients with chronic pancreatitis, ophthalmoscopic and retinal-function evaluation was performed in 28 patients with chronic pancreatitis and 19 healthy subjects. The final threshold of dark adaptation was significantly increased 40 per cent (P less than 0.001) in patients with pancreatitis, whether or not they had steatorrhea. Patients with steatorrhea had a significant decrease of about 42 per cent (P less than 0.001) in the b-wave of the electroretinogram, a measure of both rod and cone function. Seven of the 28 patients complained of difficulty with hight vision; six of these seven had morphologic lesions on ophthalmoscopic examination, confirmed by fluorescein angiography. No correlation was found between any of the retinal abnormalities and the serum vitamin A or zinc levels or glucose tolerance. Non-diabetic retinal lesions and retinal-function abnormalities are common in patients with chronic pancreatitis, even in the absence of steatorrhea.

Adult↗