Biomedical subjects
Publications and source records attributed to R Levenson.
The NHS needs to fill vacancies; the need to regenerate communities should be addressed jointly with local groups and organisations. There is much that can be done now by the NHS and its partners. A strategic approach and sustainable funding are necessary to ensure permanent change.
Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction.
We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor-FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.
Explore the source record for details and available documents.
We have identified a cohort of zebrafish expressed sequence tags encoding eight Na,K-ATPase alpha subunits and five beta subunits. Sequence comparisons and phylogenetic analysis indicate that five of the zebrafish alpha subunit genes comprise an alpha1-like gene subfamily and two are orthologs of the mammalian alpha3 subunit gene. The remaining alpha subunit clone is most similar to the mammalian alpha2 subunit. Among the five beta subunit genes, two are orthologs of the mammalian beta1 isoform, one represents a beta2 ortholog, and two are orthologous to the mammalian beta3 subunit. Using zebrafish radiation hybrid and meiotic mapping panels, we determined linkage assignments for each alpha and beta subunit gene. Na,K-ATPase genes are dispersed in the zebrafish genome with the exception of four of the alpha1-like genes, which are tightly clustered on linkage group 1. Comparative mapping studies indicate that most of the zebrafish Na,K-ATPase genes localize to regions of conserved synteny between zebrafish and humans. The expression patterns of Na,K-ATPase alpha and beta subunit genes in zebrafish are quite distinctive. No two alpha or beta subunit genes exhibit the same expression profile. Together, our data imply a very high degree of Na,K-ATPase isoenzyme heterogeneity in zebrafish, with the potential for 40 structurally distinct alpha/beta subunit combinations. Differences in expression patterns of alpha and beta subunits suggest that many of the isoenzymes are also likely to exhibit differences in functional properties within specific cell and tissue types. Our studies form a framework for analyzing structure function relationships for sodium pump isoforms using reverse genetic approaches.
Insulin stimulates K(+) uptake and Na(+) efflux via the Na(+)-K(+) pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic alpha-subunit of the Na(+)-K(+) pump. To analyze specifically the effect of insulin on the alpha(1)-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na(+)-K(+) pump alpha(1)-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of alpha(1)-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of alpha(1)-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface alpha-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of alpha(1) (at serine-18). These results suggest that insulin regulates the Na(+)-K(+) pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of alpha(1)-subunits via a signaling pathway involving PI 3-kinase and PKC.
OBJECTIVES: This study evaluated the effectiveness of registry-driven, community-based outreach directed toward children with immunization delays. METHODS: A sample of 1,856 children aged 6 to 10 months was randomly assigned to receive either outreach or no intervention. RESULTS: Children in the outreach group were more likely to receive an immunization during the observation period than children in the control group (61% vs 43%). Outreach was most effective for children with multiple risks, as measured by their immunization record; it was not effective for children whose mothers had received inadequate prenatal care. CONCLUSIONS: Registry-driven outreach can effectively identify high-risk children and bring them to care.
We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.
The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.
We have generated a stable cell line expressing FLAG epitope-tagged D3 dopamine receptors and used this cell line to study D3 receptor-protein interactions. To analyze protein interactions, we separately introduced into the stable cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A combination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf heterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 receptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG-tagged D3 receptors, suggesting that D3 receptors are capable of forming homodimers. Epitope-tagged D3nf polypeptides exhibited a markedly different cellular distribution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG-tagged D3 receptors, D3nf exhibited a punctate perinuclear distribution. When D3nf was introduced into the stable D3-expressing cell line, D3 receptors were no longer visualized at the plasma membrane. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localization. Using the HA-specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the existence of physical interaction between D3 and D3nf. This interaction appears to result in the mislocalization of D3 receptors from the plasma membrane to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.
OBJECTIVE: 1) To describe the postexposure effectiveness of varicella vaccine in a homeless shelter; and 2) to demonstrate an effective public health intervention and its implications. DESIGN: A prospective observational study. SETTING: A women and children's shelter in Philadelphia with 2 cases of varicella before intervention. OUTCOME MEASURES: Varicella in vaccinated and unvaccinated shelter residents; vaccine effectiveness for prevention of varicella when administered after exposure among children <13 years of age. RESULTS: Sixty-seven shelter residents received varicella vaccine after exposure, including 42 children <13 years of age. One child who was unvaccinated developed varicella, but no vaccinated child developed typical disease. Vaccine effectiveness was 95.2% (95% CI, 81.6%-98.8%) for prevention of any disease and 100% for prevention of moderate or severe disease among the children <13 years of age. CONCLUSION: When used within 36 hours after exposure to varicella in a setting where close contact occurred, varicella vaccine was highly effective in preventing further disease. This study provides support for the recent recommendation by the Advisory Committee on Immunization Practices to administer varicella vaccine after exposure: this practice should minimize the number of moderate or severe cases of disease and prevent prolonged outbreaks.
We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.
Explore the source record for details and available documents.
A written agreement on responsibilities for home care between local authorities, a health authority and a community trust has been welcomed by staff affected. But staff believe a written agreement is no substitute for good personal communication between different groups. Staff taking part in an evaluation of the agreement expressed concern at elderly people refusing help because of charging.
We have analyzed the role of N-linked glycosylation in functional cell surface expression of the D1 and D5 dopamine receptor subtypes. Treatment of transfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosaccharide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the plasma membrane localization of the D1 receptor. Polymerase chain reaction mutagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of mutant receptors indicated that glycosylation of residue N7 was the major determinant of D5 receptor plasma membrane localization. Mutation of a comparable site in the D1 receptor at position N5 had no effect on the delivery of the D1 receptor to the cell surface. Tunicamycin treatment during receptor biosynthesis, but not N-glycosidase F digestion of mature receptors, abrogated binding of the D5 receptor antagonist [(3)H]SCH23390, suggesting that while oligosaccharide moieties play a key role in the cell surface expression of D5 receptors, they do not appear to contribute to the receptor's ligand binding properties. Together, our data indicate a differential requirement for N-linked glycosylation in functional cell surface expression of D1 and D5 dopamine receptors.
We have used expression of chimeras between the structurally related Na,K- and H,K-ATPase alpha subunits to localize regions that determine Na,K-ATPase activity. Segments of the rat Na,K-ATPase alpha1 subunit were replaced by the corresponding portions of the rat gastric H,K-ATPase alpha subunit, and the constructs were transfected into ouabain-sensitive human HEK 293 cells. Using the ability to transfer ouabain resistance as a measure of sodium pump activity, we identified segments within the sodium pump that could be replaced with proton pump sequences without the loss of biological activity. These functionally interchangeable segments encompassed approximately 75% of the amino acid differences between the two transporters. Segments that could not be exchanged mapped to three discrete regions. One region spans residues 63-117 and includes the first transmembrane (TM) segment and a portion of the amino-terminal cytoplasmic domain. The second, from residue 320 to residue 413, encompasses TM 4 and a portion of the third cytoplasmic domain, while the third region (encompassing residues 735-861 and 898-953) includes several TM domains in the carboxyl-terminal portion of the ATPase. Our results suggest that functional differences between Na,K- and H,K-ATPase, including differences in ion transport specificity, are likely to reside within these noninterchangeable segments.
We have cloned and characterized the Na,K-ATPase beta 3 subunit gene (ATP1B3), and a beta 3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The beta 3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the beta 3 subunit gene is identical to that of the Na,K-ATPase beta 3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse beta 3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the beta 3 subunit gene are likely to be located within this conserved region. Dot blot analysis of beta 3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of beta 3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22-->23 region of Chromosome (Chr) 3, and the beta 3 pseudogene to the p13-->15 region of Chr 2.
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.