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R Levenson

Publications and source records attributed to R Levenson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells.

We examined the effects of thromboxane A2 (TxA2) on the activities of the plasminogen-plasmin system in glomerular mesangial cells. When mesangial cells are exposed to the TxA2 agonist U-46619, a substantial increase in production of plasminogen activator inhibitor-1 (PAI-1) protein is observed that is significantly greater than that induced by 10% serum alone. This increase in PAI-1 protein production is accompanied by an increase in steady-state levels of PAI-1 mRNA. This stimulation is specifically mediated by TxA2 (thromboxane prostanoid, TP) receptors, since U-46619 also stimulates PAI-1 expression in cells that are transfected with TP receptors, and this stimulation of PAI-1 production is completely blocked by the TxA2 receptor antagonist, SQ-29,548. Despite the increase in PAI-1 production, there was net stimulation of plasmin activity in the medium of mesangial cells that had been exposed to U-46619. Furthermore, U-46619 also caused an increase in tissue plasminogen activator (tPA) mRNA levels. Thus TxA2 stimulates the production of PAI-1 and plasminogen activators by mesangial cells through a receptor-dependent mechanism. In inflammatory renal diseases, the balance of these effects may modulate glomerular thrombosis and renal fibrosis.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Public health. Leading questions.

The arguments in favour of the public health director being a doctor remain powerful. It is difficult to imagine someone without a medical qualification being accepted by junior doctors, consultants and GPs. However, other disciplines may have a better understanding of social and economic determinants of health, and more skills in working with communities. Given current prevailing attitudes, it is unlikely that a professional other than a doctor could gain the confidence of the range of groups that a public health director must work with. But attitudes could and possibly should, change.

Attitude to Health↗
PubMed1997

The molar ratios of alpha and beta subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle.

The Na+-K+-ATPase consists of alpha and beta subunits proposed to function as an alpha-beta heterodimer. Skeletal muscle is characterized by expression of alpha1, alpha2, beta1, and beta2 subunit isoforms. The relative molar proportions of each subunit or each protein isoform are not known, yet their subcellular distribution and expression in muscles of different fiber types are markedly different. In this study, the molar ratio of each pump subunit isoform was measured in purified membranes from skeletal muscle and compared with those in kidney and brain microsomes. Recombinant proteins were used as standards to quantitate each isoform by immunoblotting in combination with measurements of [3H]ouabain binding. The results indicate that in kidney microsomes, which express predominantly alpha1 and beta1 isoforms, the alpha:beta subunit molar ratio is approximately 1:1. In brain microsomes, the sum of all alpha (alpha1, alpha2, and alpha3) and all beta (beta1 and beta2) subunits also yielded a molar ratio of approximately 1:1. In contrast, in red (oxidative) skeletal muscles, the all alpha:beta subunit ratio was 0.2 in plasma membranes and 0.4 in intracellular membranes. The ratio of alpha2 subunits to alpha1 subunits ranged from 1.6 in surface membranes to up to 7 in internal membranes, while the beta1 subunits exceeded the beta2 subunits by approximately 4-fold in all membrane fractions. Thus, intracellular membranes of red skeletal muscles contain primarily alpha2 and beta1 subunits. When these intracellular membranes were further subfractionated by velocity gradient centrifugation, the alpha2:beta1 subunit ratio was 0.5 in the faster migrating (larger) membranes and 1.0 in the slower migrating (smaller) ones. This was due to a progressive decrease in abundance of the beta1 subunits without a change in the concentration of alpha2 subunits per unit protein. The Na+-K+-ATPase hydrolytic activity was higher in the larger vesicles than in the smaller ones along the sucrose gradient. These results suggest that the ratio of beta to alpha subunits may serve to regulate the catalytic activity of the Na+-K+-ATPase in skeletal muscle.

Animals↗
PubMed1997

1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity.

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase beta 1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in beta 1 subunit expression occur in the absence of a detectable increase in expression of any of the three alpha subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the beta subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of beta subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the beta subunit to the plasma membrane may be independent of its binding to the alpha subunit.

Animals↗
PubMed1997

Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta.

Amino acids N886-A911 of the rat Na+-K+-ATPase alpha3-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit. The chimera (NGH26) was expressed in yeast with the rat Na+-K+ -ATPase beta1-subunit (rbeta1), the rat H+-K+-ATPase beta-subunit (HKbeta), the chimeric beta-subunit NHbeta1 (containing the carboxy-terminal ectodomain of HKbeta), or the chimeric beta-subunit HNbeta1 (containing the carboxy-terminal ectodomain of rbeta1). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKbeta and NHbeta1 rather than with rbeta1 or HNbeta1. Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKbeta or NHbeta1. These results suggest that the sequence Q905-V930 interacts with the HKbeta-subunit on the extracellular side of the cell membrane. The NGH26 + HKbeta complex is less stable than alpha3 + HKbeta when heated and also has a lower binding affinity for ouabain [dissociation constant (Kd) = 63 nM] compared with alpha3 + rbeta1 or alpha3 + HKbeta (K(d) = 5-10 nM). In contrast, the NGH26+NHbeta1 complex is thermally as stable as alpha3 + rbeta1 complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type. These results indicate that the amino acids Q905-V930 of the rat gastric H+-K+-ATPase alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.

Amino Acid Sequence↗
PubMed1997

PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone.

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.

1-Methyl-3-isobutylxanthine↗
PubMed1997

A program to immunize hospitalized preschool-aged children: evaluation and impact.

OBJECTIVE: The Standards for Pediatric Immunization Practices suggest that hospitalization be viewed as an opportunity to vaccinate children. The purpose of the present study is 1) to determine the immunization status of an urban population of hospitalized preschool-aged children, 2) to study the impact of an immunization program designed to vaccinate hospitalized 0 to 2-year-old children who are underimmunized at admission, and 3) to make immunization a routine part of care for the hospitalized child. METHODS: Prospective evaluation of the immunization status of hospitalized 0 to 2-year-old residents of Philadelphia admitted to an urban children's hospital was performed. With verification of the child's immunization record through the primary care provider (PCP), needed immunizations were given and records were forwarded to notify the PCP. Educational information was provided to families and health care providers. MAIN OUTCOME MEASURE: The percentage of children fully immunized on admission compared with the percentage at the time of discharge. Results. Two thousand three hundred twenty-nine children from 0 to 2 years of age were hospitalized during the 22-month study period. Immunization records were verified in 86% (2006), requiring an average of 1.5 phone calls to the PCP. The mean patient age was 10 months. Average hospital length of stay was 4 days. On admission, 49% (980) of the 2006 study patients were fully immunized. The remaining 51% (1026) were eligible for vaccination. Immunizations were delayed greater than or equal to 2 months in 18% (355) of the children. Neither type of health care insurance nor site of primary care affected the immunization status of those evaluated at the time of admission. Sixty-six percent (N = 674) of eligible patients received at least one vaccination before hospital discharge. Medical contraindications accounted for only 4% of the reasons eligible patients were not immunized. Of the 2006 children evaluated, the percentage of those fully vaccinated for age increased significantly from 44% on admission to 70% on discharge. CONCLUSION: As a result of this program, there was a significant improvement in vaccination percentage at the time of hospital discharge in this group of urban preschool-aged children. The development of an immunization program to vaccinate hospitalized preschool children is an opportunity to immunize in the urban setting where there is a high prevalence of underimmunization. In addition, it provides an opening for educational programs for families, nurses, and housestaff and linkage to the community PCPs.

Child, Preschool↗
PubMed1996

Localization of cytoplasmic and extracellular domains of Na,K-ATPase by epitope tag insertion.

We have used epitope tag addition to analyze the transmembrane topology of the Na,K-ATPase catalytic (alpha) subunit. An antigenic peptide derived from the hemagglutinin (HA) of influenza virus was inserted at 15 different positions within the rat Na,K-ATPase alpha 1 subunit isoform. The functional integrity of the tagged proteins was tested by their capacity to confer ouabain resistance upon human HEK 293 cells. Constructs with the tag at aa positions 119, 173, 318, 815, 881, 953, 987, and 1023 conferred ouabain resistance, and the mutant proteins were detectable in the plasma membrane of transfected cells. In contrast, alpha 1 subunits with insertions at aa positions 338, 797, 805, 868, 895, 910, and 921 were unable to confer drug resistance. Immunofluorescence analysis of permeabilized and intact cells using a monoclonal antibody specific for the HA epitope showed that double tags at positions 119 and 318 were located extracellularly, whereas single or double tags at positions 173, 815, 881, 987, and 1023 were cytoplasmically disposed. These results are consistent with an eight transmembrane domain arrangement for the alpha subunit. Epitope insertion within TM4, and the region linking transmembrane segments TM6-TM7, caused the loss of alpha subunit function, suggesting that the integrity of these domains is essential for the proper biosynthesis and/or maturation of the alpha subunit.

Animals↗
PubMed1996

Identification of the mammalian Na,K-ATPase 3 subunit.

We have isolated and characterized cDNA clones encoding the human and rat Na,K-ATPase beta3 subunit isoform. The human cDNA encodes a polypeptide of 279 amino acids that exhibits primary sequence and secondary structure similarities to Na,K-ATPase beta subunit isoforms. Sequence comparisons showed that the human beta3 subunit closely resembles the beta3 subunit of Xenopus laevis (59% amino acid identity) and is less similar to the human Na,K-ATPase beta1 and beta2 subunits (38% and 48% amino acid identity, respectively). By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the beta3 subunit gene to murine chromosome 7. Northern blot analysis revealed that the beta3 subunit gene encodes two transcripts that are expressed in a variety of rat tissues including testis, brain, kidney, lung, stomach, small intestine, colon, spleen, and liver. Identification of the mammalian beta3 subunit suggests an even greater potential for Na,K-ATPase isoenzyme diversity than previously realized.

Amino Acid Sequence↗
PubMed1996

A new sodium channel alpha-subunit gene (Scn9a) from Schwann cells maps to the Scn1a, Scn2a, Scn3a cluster of mouse chromosome 2.

We have used a total of 27 AXB/BXA recombinant inbred mouse strains to determine the chromosomal location of a newly identified gene encoding an alpha-subunit isoform of the sodium channel from Schwann cells, Scn9a. Linkage analysis established that Scn9a mapped to the proximal segment of mouse chromosome 2. The segregation of restriction fragment length polymorphisms in 145 progeny from a Mus spretus x C57BL/6J backcross indicates that Scn9a is very tightly linked to Scn1a (gene encoding the type I sodium channel alpha-subunit of the brain) and forms part of a cluster of four Scna genes located on mouse chromosome 2.

Animals↗
PubMed1996

Localization of dopamine D4 receptors in GABAergic neurons of the primate brain.

Dopamine receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five mammalian dopamine-receptor subtypes, the D4 subtype is of particular interest because of its high affinity for the atypical neuroleptic clozapine. Interest in clozapine stems from its effectiveness in reducing positive and negative symptoms in acutely psychotic and treatment-resistant schizophrenic patients without eliciting extrapyramidal side effects. We have produced a subtype-specific antibody against the D4 receptor and localized it within specific cellular elements and synaptic circuits of the central nervous system. The D4-receptor antibody labelled GABAergic neurons in the cerebral cortex, hippocampus, thalamic reticular nucleus, globus pallidus and the substantia nigra (pars reticulata). Labelling was also observed in a subset of cortical pyramidal cells. Our findings suggest that clozapine's beneficial effects in schizophrenia may be achieved, in part, through D4-mediated GABA modulation, possibly implicating disinhibition of excitatory transmission in intrinsic cortical, thalamocortical and extrapyramidal pathways.

Animals↗
PubMed1996

Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence.

P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

Animals↗
PubMed1995

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Alternative Splicing↗
PubMed1995

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

Amino Acid Sequence↗
PubMed1995

Membrane topology of P-glycoprotein as determined by epitope insertion: transmembrane organization of the N-terminal domain of mdr3.

P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR). Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain. To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376). Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells. Intracellular and extracellular localization of the tag in the full-length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope. While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance. The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane. These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions. These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6). Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

A Russian version of SNOMED-International.

Medical information systems are becoming widely used in Russia--most of them based on small- and medium-sized networks of PCUs running xBASE software. The major difficulty facing these systems is not the availability of adequate hardware and software, but rather the absence of standard terminology. The only international standard currently in use is the Russian translation of ICD-9. Development, adoption, and promotion of other international classifications and nomenclatures could remedy this situation. SNOMED-International [1] appeared to be well-suited for adaptation to the Russian medical environment because of its multiaxial structure and rich terminology. Its use could also facilitate international as well as national data collection and cooperative studies, and it may find its first application in a planned correlation of toxicology and autopsy data from the Leningrad region. Since the complete SNOMED-International contains more than 130,000 terms, the first phase of this project is restricted to the translation of the SNOMED-International Microglossary, a subset of 12,500 pathology-related diagnoses and descriptive terms. Each term was given up to three Russian equivalents which were entered in a database. Then all the Russian terms were printed, edited, and their corrections were entered in the same database. This core set of the Russian terms will be continuously updated and expanded as it is used in actual coding applications. The procedure will be as follows: if a phrase is encountered that could not be mapped to a term in the current version of the Russian nomenclature, the complete SNOMED lexicon will be searched manually for an English equivalent, its code applied to a Russian term, and this term will then be added to the Russian SNOMED database. This approach has the advantage that most of the terms in the Russian nomenclature will not be apriori translations from English, but will arise from the original texts found in real documents. This project is now in progress; the translation of the SNOMED-International Microglossary has been completed. The more difficult task of developing procedures for parsing free text sentences and updating the nomenclature is under development.

Russia↗