PubMed Health⌕ Search

Biomedical subjects

J R Casey

Publications and source records attributed to J R Casey.

30 records · Page 2Linked to original sources
PubMed1994

AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia.

Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH-terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Müller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Müller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10-15, correlating strongly with the onset of retinal function.

Aging↗
PubMed1994

Acute splenic sequestration in the absence of palpable splenomegaly.

PURPOSE: Acute splenic sequestration in children with sickle hemoglobinopathies is a medical emergency characterized by splenomegaly, anemia, and thrombocytopenia. PATIENTS AND METHODS: We describe a young girl with hemoglobin SC disease who experienced an acute splenic sequestration without palpable splenomegaly. CONCLUSIONS: We emphasize the need for a high index of suspicion for splenic sequestration and the utility of ultrasonography in documenting splenomegaly.

Acute Disease↗
PubMed1993

Molecular characterization of the band 3 protein from Southeast Asian ovalocytes.

Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocytosis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codons 400-408, encoding a 9-amino-acid sequence located at the boundary between the cytosol and the first transmembrane segment in Band 3, the erythrocyte anion transport protein. We have carried out an extensive characterization of Band 3 isolated from SAO erythrocytes which contain about 50% mutant Band 3. A slightly higher proportion of Band 3 in SAO erythrocytes was left associated with the cytoskeleton after extraction of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 contained a higher proportion of tetramers relative to dimers (50% tetramer) than normal Band 3 (33% tetramer). The circular dichroism spectrum of Band 3 from SAO erythrocytes was very similar to the spectrum for normal Band 3. Enzymatic deglycosylation and tomato lectin binding showed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dramatic alteration in the inhibitor binding site. In conclusion, deletion of 9 amino acids from Band 3 on the cytosolic side of the membrane affects the properties (glycosylation and inhibitor binding) of Band 3 on the opposite side of the membrane without dramatic changes in the secondary and quaternary structure of the protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1993

Detergent interaction with band 3, a model polytopic membrane protein.

The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties.

The structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation. Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

Laparoscopic cholecystectomy in young patients with sickle hemoglobinopathies.

Nine young patients with sickle hemoglobinopathies successfully underwent laparoscopic cholecystectomy; no complications resulted from the procedure. The mean postoperative hospital stay was 1.6 days. This technique appears to be a safe and efficacious procedure in children with sickle hemoglobinopathies who require cholecystectomy for cholelithiasis.

Adolescent↗
PubMed1991

Analysis of the oligomeric state of Band 3, the anion transport protein of the human erythrocyte membrane, by size exclusion high performance liquid chromatography. Oligomeric stability and origin of heterogeneity.

The oligomeric state of human Band 3 (Mr = 95,000), the erythrocyte membrane anion exchanger, was examined by size exclusion high performance liquid chromatography in solutions containing the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). Band 3 was heterogeneous with respect to oligomeric composition, the predominant (70%) species being a dimer that bound 0.57 mg of C12E8/mg of protein (Stokes radius = 78 A, s20,w = 6.9 S). Variable amounts of larger oligomers were also present; however, no evidence for equilibration between oligomeric species was observed in detergent solution. Analytical and large zone size exclusion chromatography showed that Band 3 could not be dissociated to monomers, other than by protein denaturation. The membrane domain of Band 3 (Mr = 52,000) was also dimeric, but without evidence for higher oligomeric forms, which implies that the interactions responsible for higher associations involve the cytoplasmic domain. Prelabeling of Band 3 with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate had no effect upon the oligomeric state of either intact Band 3 or its 52-kDa membrane domain. Band 3 oligomeric state could be reversibly changed in the membrane by altering the pH of the solution. The fraction of Band 3 not associated with the cytoskeleton was almost entirely dimeric. Band 3 purified from erythrocytes separated by density gradient centrifugation revealed that older red cells contained a larger proportion of higher oligomers than did younger cells. We conclude that Band 3, in the membrane and in C12E8 solution, exists as a mixture of dimers and larger oligomers. The higher oligomers interact with the cytoskeleton, increase in amount with cell age, and are held together by interactions of the cytoplasmic domain.

Anion Exchange Protein 1, Erythrocyte↗
PubMedDate not supplied

Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein.

4,4'-Diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS), a bifunctional inhibitor of anion exchange in erythrocytes, reacts with Lys-539 in band 3 at neutral pH and crosslinks to Lys-851 at alkaline pH. The accessibility of H2DIDS-labelled band 3 was determined using an anti-H2DIDS antibody and proteolysis. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that a polyclonal antibody raised against H2DIDS-labelled keyhole limpet hemocyanin bound a variety of stilbene disulphonates in the following order of affinities, H2DIDS having the highest affinity: H2DIDS > 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) > 4-acetamido-4'-isothiocyanostilbene-2,2'disulphonate (SITS) > 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) > 4,4'-diaminostilbene-2,2'-disulphonate (DADS). The antibody readily detected mono- or bifunctionally H2DIDS-labelled band 3 and proteolytic fragments on immunoblots. H2DIDS attached to Lys-539 is retained in a 7.5 kDa membrane-associated peptide after papain treatment of ghost membranes while the sequence around Lys-851 is more accessible. The band 3 proteolytic fragments protected by the membrane from proteolysis remained associated as a specific complex with a Stokes radius slightly smaller than the dimeric membrane domain after solubilization in detergent solution and retained 82% of the amino acid content of the membrane domain. Circular dichroism (CD) measurements of this H2DIDS-labelled complex showed that it had a very high helical content (86%). The loops connecting the transmembrane segments in H2DIDS-labelled band 3 are therefore not required to maintain transmembrane helix-helix interactions. Denatured band 3 prelabelled with H2DIDS was more readily immunoprecipitated with the anti-H2DIDS antibody than was native band 3 in detergent solution. Deglycosylation of band 3 or proteolytic cleavage of the extramembranous loops did not enhance immunoprecipitation of H2DIDS-labelled band 3. The stilbene disulphonate inhibitor site is therefore relatively inaccessible and is bound by a bundle of helices in the native band 3 protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

Successful use of extracorporeal membrane oxygenation in the treatment of acute chest syndrome in a child with severe sickle cell anemia.

Extracorporeal membrane oxygenation (ECMO) is widely used in the treatment of respiratory and cardiovascular failure in neonatal patients. The authors present a case of a child with hemoglobin SS disease who was treated with ECMO after acute chest syndrome and acute respiratory distress syndrome developed. They also present data from the Extracorporeal Life Support Organization on this use of ECMO from other centers. To date, there have been 15 pediatric patients with acute chest syndrome treated with ECMO. Survival rate has been 26%. In selected patients with severe disease, ECMO can provide support at a lower mean airway pressure, allow for aggressive pulmonary lavage, and maintain adequate tissue oxygen delivery until the patient is more stable. Patients who might benefit include those with poor ventilation secondary to mucous plugging and barotrauma. The best success with these patients might be anticipated from venoarterial ECMO. Patients with severe cardiac or neurologic deterioration may constitute a group less likely to survive.

Anemia, Sickle Cell↗