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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
PubMed2005

Accuracy of GDx variable corneal compensation polarization measurements in normal human eyes: effect of accommodation, cycloplegia, focus, pupil size, and eye selection on reproducibility.

PURPOSE: To evaluate the reproducibility of variable corneal compensation (VCC) and the effect of accommodation, cycloplegia, eye selection, focus, and pupil size on this measurement of polarization. METHODS: Using a GDx scanning laser polarimeter, multiple measurements of the VCC were obtained from each eye of 33 healthy, young adults under differing conditions. Pupil size and refraction were independently measured with a pupillometer and an autorefractometer. The effects of eye, instrument focus, pupil diameter, cycloplegia, and accommodation were statistically assessed. RESULTS: The reproducibility of a single retardation measurement and its axis, as determined by the standard deviations (SD) of repeated measurements, is +/-1.58 nm and 2.10 degrees , respectively. There is a difference in retardation between right and left eyes, of 5.26+/-9 nm, P=0.002. Increasing pupil size increases retardation. Cycloplegia or defocusing decreases retardation, and pharmacologically induced accommodation has no effect on retardation. CONCLUSIONS: The retardation and its axis are highly reproducible measurements when the pupil is of physiologic size and the GDx is properly focused. There is a consistent difference in VCC retardation between the paired right and left eyes. This difference may reflect equipment-induced measurement artefact and/or an anatomic asymmetry between the paired eyes of the subjects studied. Clinicians should be cautious when comparing interocular VCC measurements between paired right and left eyes and using data pooled from both eyes for age-adjusted, normalized standards.

Accommodation, Ocular↗
PubMed2001

Structure-function analysis of the antioxidant properties of haptoglobin.

Haptoglobin serves as an antioxidant by virtue of its ability to prevent hemoglobin-driven oxidative tissue damage. It was recently demonstrated that an allelic polymorphism in the haptoglobin gene is predictive of the risk for numerous microvascular and macrovascular diabetic complications. Because these complications are attributed in large part to an increase in oxidative stress, a study was conducted to determine whether the different protein products of the 2 haptoglobin alleles differed in the antioxidant protection they provided. A statistically significant difference was found in the antioxidant capacity of purified haptoglobin protein produced from the 2 different alleles, consistent with the hypothesis that differences in genetically determined antioxidant status may explain differential susceptibility to diabetic vascular complications. These differences may be amplified in the vessel wall because of differences in the sieving capacity of the haptoglobin types. Therefore, an attempt was made to identify the minimal haptoglobin sequences necessary to inhibit oxidation by hemoglobin in vitro, and 2 independent haptoglobin peptides that function in this fashion as efficiently as native haptoglobin were identified. Identification of the biochemical basis for differences among haptoglobin types may lead to the rational development of new pharmacologic agents, such as the mini-haptoglobin described here, to avert the development of diabetic vascular complications.

Alleles↗
PubMed2000

Hypoxic induction of vascular endothelial growth factor is markedly decreased in diabetic individuals who do not develop retinopathy.

OBJECTIVE: A small percentage of patients do not develop any evidence of diabetic retinopathy (DR) even after many years of diabetes. Vascular endothelial growth factor (VEGF) has been implicated in the development of DR. Therefore, we sought to determine if the regulation of VEGF differs in those patients who develop DR after many years of diabetes compared with those who do not develop DR. RESEARCH DESIGN AND METHODS: We performed standard 7-field stereoscopic fundus photography on 95 consecutive patients with type 1 and type 2 diabetes. Patients were categorized into 3 groups according to the presence or absence of DR and the duration of diabetes: group 1 was defined by presence of DR, group 2 was defined by absence of DR after >10 years duration of diabetes, and group 3 was defined by absence of DR with long-standing diabetes (> or =20 years for type 1 diabetes and > or =15 years for type 2 diabetes). Monocytes from 40 ml peripheral blood were isolated from all patients, and the hypoxic induction of VEGF was determined in vitro. RESULTS: We found no significant difference in the basal level of VEGF in patients with and without DR. However, we did find a markedly significant difference in the hypoxic induction of VEGF between patients from group 1 and group 3 (4.35+/-0.55 vs. 1.87+/-0.3, P<0.00013). CONCLUSIONS: This study suggests that 1 mechanism for the absence of DR in patients with long-standing diabetes is a decreased hypoxic induction of VEGF.

Cell Hypoxia↗
PubMed1998

Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose expression is dramatically induced by hypoxia due in large part to an increase in the stability of its mRNA. Here we show that HuR binds with high affinity and specificity to the element that regulates VEGF mRNA stability by hypoxia. Inhibition of HuR expression abrogates the hypoxia-mediated increase in VEGF mRNA stability. Overexpression of HuR increases the stability of VEGF mRNA. However, this only occurs efficiently in hypoxic cells. We further show that the stabilization of VEGF mRNA can be recapitulated in vitro. Using an S-100 extract, we show that the addition of recombinant HuR stabilizes VEGF mRNA markedly. These data support the critical role of HuR in mediating the hypoxic stabilization of VEGF mRNA by hypoxia.

Antigens, Surface↗
PubMed1997

Sequencing of the human vascular endothelial growth factor (VEGF) 3' untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose mRNA expression is induced by hypoxia. This induction is due in large part to an increase in the stability of its mRNA. The RNA sequences and cognate proteins responsible for this increased stability with hypoxia are not well understood. In order to identify regions of functional importance in the 3'UTR of VEGF mRNA, we have sequenced the human VEGF 3'UTR and compared it to the rat sequence. Overall sequence homology was 82% with complete conservation of all four potential polyadenylation signals and both nonameric instability elements. Five hypoxia-inducible RNA protein-binding (HI-RPB) sites were identified by RNA electromobility shift assay (EMSA) in the human and rat genes. EMSA and competition studies suggest that these sites bind a similar or related protein complex. On average, the five sites were 95% conserved at the nucleotide level between the rat and corresponding human sequence. This conservation taken together with several previously described, independent correlations between the presence of these RNA-protein complexes and an increase in VEGF mRNA stability suggest an important functional role for these sites in mediating hypoxia-inducible VEGF mRNA stability.

Animals↗
PubMed1997

Ocular absorption, blood levels, and excretion of dorzolamide, a topically active carbonic anhydrase inhibitor.

Dorzolamide is a powerful inhibitor of carbonic anhydrase (CA) II that penetrates the sclera and cornea to reach the ciliary process and lowers formation of HCO3 and aqueous humor. The usual dose applied to the eye in treatment of glaucoma is 1 drop (30 microL of 2% solution) every 8 hr to each eye, or a total daily dose of 4 mg. On this regime, the red cells accumulated drug over a period of 8 days, reaching a value of 20-25 microM, which corresponds to the concentration of CA II in human red cells. This drug concentration persisted throughout the 18 months of application. The plasma concentration was 0.034 microM, or 1/700 that of the red cells. This plasma concentration corresponds to that calculated from the dilution of administered drug into body water. The data are well fitted into the equilibrium expression for KI of dorzolamide against CA II at 37 degrees C, as 8 x 10(-9) M. The red cells also contain a small amount (5 microM) of the N-des-ethyl metabolite, probably reflecting its modest binding to CA I. In the initial 8-day drug period, virtually none appeared in the urine since CA II sites were being filled. At steady state, renal excretion was 1.3 mg/day and the renal clearance 90 ml/min. These excretion numbers include the small (20%) amount of the des-ethyl metabolite of dorzolamide. The relation of these data to lowering of intraocular pressure is clear. By the systemic route, an inhibitor such as acetazolamide is effective when free drug concentration in plasma is 2.5 microM. In the case of topical drugs, as shown here, the plasma concentration is some 100 x lower, but the concentration in ciliary process is 2-10 microM, comparable to that following systemic drugs (1). In conclusion, the concentration in plasma (reflecting free drug) of dorzolamide is about 1/200 of that needed for systemic effects as seen following acetazolamide or methazolamide. Thus, there is a clear pharmacological basis for the lack of any physiological effects of ocular dorzolamide, except on the eye itself.

Administration, Topical↗
PubMed1997

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascularization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/1 (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/l (383 ng/m1) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/l increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.

Animals↗
PubMed1996

Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein.

Hypoxia induces an increase in the stability of the mRNA encoding vascular endothelial growth factor (VEGF). We have previously demonstrated that a 500-base region of the 3'-untranslated region of VEGF mRNA that is critical for stabilization of VEGF mRNA in an in vitro degradation assay forms a RNA-protein complex in a hypoxia-inducible fashion. We report here the identification of three adenylate-uridylate-rich RNA elements within this region that form an identical or closely related hypoxia-inducible RNA-protein complex. This complex is constitutively elevated in a tumor cell line lacking the wild type von Hippel-Lindau tumor suppressor gene and in which VEGF mRNA is constitutively stabilized. Furthermore, the glucose transporter-1 mRNA, which is also stabilized by hypoxia, forms a hypoxia-inducible RNA-protein complex with similar sequence and protein binding characteristics to that described for VEGF mRNA. Finally, RNA affinity purification and UV cross-linking were used to identify three proteins of 32, 28, and 17 kDa that are derived from this hypoxia-inducible RNA-protein complex.

Adenine↗
PubMed1996

Post-transcriptional regulation of vascular endothelial growth factor by hypoxia.

The major control point for the hypoxic induction of the vascular endothelial growth factor (VEGF) gene is the regulation of the steady-state level of the mRNA. We previously demonstrated a discrepancy between the transcription rate and the steady-state mRNA level induced by hypoxia. This led us to examine the post-transcriptional regulation of VEGF expression. Actinomycin D experiments revealed that hypoxia increased VEGF mRNA half-life from 43 +/- 6 min to 106 +/- 9 min. Using an in vitro mRNA degradation assay, the half-life of VEGF mRNA 3'-untranslated region (UTR) transcripts were also found to be increased when incubated with hypoxic versus normoxic extracts. Both cis-regulatory elements involved in VEGF mRNA degradation under normoxic conditions and in increased stabilization under hypoxic conditions were mapped using this degradation assay. A hypoxia-induced protein(s) was found that bound to the sequences in the VEGF 3'-UTR which mediated increased stability in the degradation assay. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the hypoxia-induced stabilization of VEGF 3'-UTR transcripts and inhibited hypoxia-induced protein binding to the VEGF 3'-UTR. These findings demonstrate a significant post-transcriptional component to the regulation of VEGF.

Animals↗
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↗