Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.     

Imaging spectrum of thrombo-occlusive vascular disease associated with antiphospholipid antibodies

The association of antiphospholipid antibodies with unexplained thrombo-occlusive vascular disease is well known but often remains unrecognized. The most well-studied clinical manifestation is venous thrombosis, but arterial occlusive disease involving multiple sites is also well documented. Twenty-six cases of thrombo-occlusive disease were observed in 22 patients over a 3-year period. Magnetic resonance imaging and angiography were used to make the diagnoses. None of the patients who underwent angiography or venography developed thrombolytic disease related to the puncture site. This group of patients with antiphospholipid antibody syndrome had a wide distribution of arterial and venous thrombotic disease. Radiologists should consider antiphospholipid antibody syndrome in the differential diagnosis when evaluating thrombo-occlusive vascular disease that is unexpected or occurs without risk factors. Knowledge of antiphospholipid antibody status has important implications for prognosis and therapy.     

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.     

Thrombolytic and endovascular treatment of peripartum iliac vein thrombosis: a case report

This case report details the multidisciplinary treatment of peripartum left iliac vein thrombosis using percutaneous catheter-directed urokinase thrombolysis and balloon thromboplasty. Enhanced chances for long-term patency and the normalization of venous function make these minimally invasive procedures accepted options for the treatment of iliofemoral deep venous thrombosis in selected peripartum patients.     

The effects of growth factors on Tenon''s capsule fibroblasts in serum-free culture

Recent studies from our laboratory have shown that in the mouse and rat nephron Ca2+ and Mg2+ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca2+ and Mg2+ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PDte.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca2+ and Mg2+ net fluxes in the mTAL are not driven by the transepithelial PDte. At zero voltage, a small but significant net secretion of Ca2+ into the tubular lumen was observed. With a high lumen-positive PDte generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca2+ and Mg2+ reabsorption was noted; instead significant and sustained Ca2+ and Mg2+ net secretion occurred. When a lumen-positive PDte was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca2+ net secretion and a lesser Mg2+ net secretion, not modified by ADH, were observed. Replacement of Na+ by K+ in the lumen perfusate induced, in the absence of PDte changes, important but reversible net secretions of Ca2+ and Mg2+. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca2+ and Mg2+ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca2+ and Mg2+, the secretory processes would appear to be of cellular origin.     

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.     

Typicality effects in attitudes toward social policies: A concept-mapping approach.

Social typicality effects occur when people apply their attitudes more consistently toward typical than toward atypical category members presumably because attitudes are directed toward the prototypic category member. Four studies tested whether individuals also apply social policy attitudes more consistently toward typical than toward atypical persons affected by the policy. Study 1 used attitude concept maps to assess how extensively individuals spontaneously reference the typical person affected by a social policy. Death penalty proponents did so more than did opponents; welfare proponents and opponents did so equally. In 3 other studies, death penalty proponents displayed greater typicality effects in sentencing "convicted murderers" than did opponents, welfare proponents and opponents displayed equal typicality effects in evaluating cases of suspected welfare fraud, and individuals whose attitude concept maps about welfare referred more to persons displayed greater typicality effects than did individuals whose maps referred more to principles. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Reliability and validity of the Gastrointestinal Symptom Rating Scale in patients with gastroesophageal reflux disease

American visceral leishmaniasis is a main public health matter in Brazil. Since dogs have been incriminated as the main urban reservoir of AVL agent Leishmania chagasi, a cohort study aimed at understanding the dynamics of the canine infection was carried out in Jequié--an endemic community in the Northeast of Brazil. The inhabited urban and periurban areas of Jequié were divided into 140 clusters of 0.25 km2. All 1681 dogs domiciled in 34 randomly selected clusters were screened for Leishmania antibodies in an enzyme-linked immunosorbent assay. After the seropositive dogs were painlessly eliminated, a cohort of 1286 seronegative dogs was followed up for 18 months, yielding a total of 1739.7 dog-years. The overall incidence of Leishmania infection, as assessed by the detection of Leishmania antibodies in blood samples collected every six months, was 6.55 cases/100 dog-years (95% confidence interval; CI 6.04-7.26). Two subsets of clusters, with 0.70 and 1.35 relative risks of infection, were identified. The annual emigration rate was 2.26 cases/100 dog-years (95% CI 1.86-2.66). The implications of these findings for the control of American visceral leishmaniasis are discussed.     

Linkage analysis of candidate genes in autoimmune thyroid disease: 1. Selected immunoregulatory genes. International Consortium for the Genetics of Autoimmune Thyroid Disease

Graves' and Hashimoto's diseases are autoimmune thyroid diseases in which the genetic contribution is complex. For this reason, identification of necessary susceptibility genes has been difficult. However, a number of immunoregulatory genes have been implicated by association studies, including: CTLA-4, a recently described protein involved in antigen presentation, located on chromosome 2q33; the T-cell receptor V alpha and V beta gene complexes, located on 14q11 and 7q35, respectively; and the Ig gene complex (IgH), located on 15q11. We used polymorphic microsatellite markers located within these genes, or gene complexes, to test for linkage (rather than association), to each of these candidates. Using markers within the loci allowed us to assume a fixed recombination fraction of 0.01 in the tested model. Three hundred eight subjects from 48 multiplex families were studied, with 142 affected subjects. Using this set of families, we have previously shown evidence of linkage with a major susceptibility locus for Graves' disease (GD-1) on 14q24.3-31, with a maximum lod (logarithm + odds) score of 2.1, at a penetrance of 80% and with a dominant mode of inheritance. In the present study, we obtained consistently negative lod scores for each of the candidate genes, assuming either dominant or recessive modes of inheritance. These data, therefore, showed evidence against linkage with all the candidate genes. Unlike association studies, linkage analyses detect major genetic influences on disease susceptibility exerted by the linked loci. The lack of linkage for the immunoregulatory genes that were studied indicated, therefore, that they were not major contributors to disease etiology.