Five-year prospective study of paediatric acute otitis media in Rochester, NY: modelling analysis of the risk of pneumococcal colonization in the nasopharynx and infection.

During a 5-year prospective study of nasopharyngeal (NP) colonization and acute otitis media (AOM) infections in children during the 7-valent pneumococcal conjugate vaccine (PCV) era (July 2006-June 2011) we studied risk factors for NP colonization and AOM. NP samples were collected at ages 6, 9, 12, 15, 18, 24, and 30 months during well-child visits. Additionally, NP and middle ear fluid (MEF) samples were collected at onset of every AOM episode. From 1825 visits (n = 464 children), 5301 NP and 570 MEF samples were collected and analysed for potential otopathogens. Daycare attendance, NP colonization by Moraxella catarrhalis, and siblings aged <5 years increased the risk of Streptococcus pneumoniae NP colonization. NP colonization with S. pneumoniae, M. catarrhalis, or Haemophilus influenzae and a family history of OM increased the risk of AOM. Risk factors that increase the risk of pneumococcal AOM will be important to reassess as we move into a new 13-valent PCV era, especially co-colonization with other potential otopathogens.

Pharmacodynamic analysis and clinical trial of amoxicillin sprinkle administered once daily for 7 days compared to penicillin V potassium administered four times daily for 10 days in the treatment of tonsillopharyngitis due to Streptococcus pyogenes in children.

An a priori pharmacokinetic/pharmacodynamic (PK/PD) target of 40% daily time above the MIC (T >MIC; based on the MIC(90) of 0.06 microg/ml for Streptococcus pyogenes reported in the literature) was shown to be achievable in a phase 1 study of 23 children with a once-daily (QD) modified-release, multiparticulate formulation of amoxicillin (amoxicillin sprinkle). The daily T >MIC achieved with the QD amoxicillin sprinkle formulation was comparable to that achieved with a four-times-daily (QID) penicillin VK suspension. An investigator-blinded, randomized, parallel-group, multicenter study involving 579 children 6 months to 12 years old with acute streptococcal tonsillopharyngitis was then undertaken. Children were randomly assigned 1:1 to receive either the amoxicillin sprinkle (475 mg for ages 6 months to 4 years, 775 mg for ages 5 to 12 years) QD for 7 days or 10 mg/kg of body weight of penicillin VK QID for 10 days (up to the maximum dose of 250 mg QID). Unexpectedly, the rates of bacteriological eradication at the test of cure were 65.3% (132/202) for the amoxicillin sprinkle and 68.0% (132/194) for penicillin VK (95% confidence interval, -12.0% to 6.6%). Thus, neither antibiotic regimen met the minimum criterion of > or =85% eradication ordinarily required by the U.S. FDA for first-line treatment of tonsillopharyngitis due to S. pyogenes. The results of subgroup analyses across demographic characteristics and current infection characteristics and by age/weight categories were consistent with the primary-efficacy result. The clinical cure rates for amoxicillin sprinkle and penicillin VK were 86.1% (216/251) and 91.9% (204/222), respectively (95% confidence interval, -11.6% to -0.4%). The results of a post hoc PD analysis suggested that a requirement for 60% daily T >MIC(90) more accurately predicted the observed high failure rates for bacteriologic eradication with the amoxicillin sprinkle and penicillin VK suspension studied. Based on the association between longer treatment courses and maximal bacterial eradication rates reported in the literature, an alternative composite PK/PD target taking into consideration the duration of therapy, or total T >MIC, was considered and provides an alternative explanation for the observed failure rate of amoxicillin sprinkle.

Correlation of higher antibody levels to pneumococcal proteins with protection from pneumococcal acute otitis media but not protection from nasopharyngeal colonization in young children.

OBJECTIVES: We previously found that nasopharyngeal (NP) colonization by Streptococcus pneumoniae elicits mucosal antibody responses to three protein vaccine candidates: pneumococcal histidine triad protein D (PhtD), pneumococcal choline-binding protein A (PcpA), and detoxified pneumolysin (PlyD1). Here we sought to determine if mucosal antibody levels to the proteins correlated with protection from acute otitis media (AOM) and NP colonization. METHODS: A total of 228 NP samples were prospectively collected from 100 healthy infants at 6-24 months of age. Whenever children were diagnosed with AOM, middle ear fluids were collected to confirm the diagnosis by microbiological culture. NP mucosal IgG and IgA were quantified by ELISA. RESULTS: Higher NP mucosal antibody levels to S. pneumoniae proteins correlated with significantly decreased likelihood of developing AOM caused by S. pneumoniae during 3 to 12 months of subsequent prospective monitoring. Specifically, children who did not experience AOM (n=111samples) caused by S. pneumoniae had two- to five-fold higher mucosal IgG levels to PcpA (all p values <0.01), six- to eight-fold higher IgA to PhtD (all p values <0.05); two- to three-folder higher IgA to PcpA (all p values <0.05), and two- to three-fold higher IgA to PlyD1 (p 0.08, p 0.03 and p 0.08) compared with children who did experience AOM (n=18samples). No association between mucosal antibody levels to the three proteins and NP colonization with S. pneumoniae was found. CONCLUSION: Higher NP mucosal IgG levels to PcpA, and IgA to PhtD, PcpA and PlyD1 correlate with reduced risk of development of S. pneumoniae AOM infection but not with reduced risk of NP colonization in young children.

Molecular basis for angiotensin II-induced increase of chloride/bicarbonate exchange in the myocardium.

Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pH(i)) by Na(+)-independent Cl(-)/HCO(3)(-) exchange. Angiotensin II (Ang II) activates protein kinase C (PKC) and increases anion exchange activity in the myocardium. Elevated anion exchange activity has been proposed to contribute to the development of cardiac hypertrophy. Our Northern blots showed that adult rat heart expresses AE1, AE2, AE3fl, and AE3c. Activity of each AE isoform was individually measured by following changes of pH(i), associated with bicarbonate transport, in transfected HEK293 cells. Exposure to the PKC activator, PMA (150 nmol/L), increased the transport activity of only the AE3fl isoform by 50+/-11% (P<0.05, n=6), consistent with the increase observed in intact myocardium. Cotransfection of HEK293 cells with AE3fl and AT1(a)-Ang II receptors conferred sensitivity of anion transport to Ang II (500 nmol/L), increasing the transport activity by 39+/-3% (P<0.05, n=4). PKC inhibition by chelerythrine (10 micromol/L) blocked the PMA effect. To identify the PKC-responsive site, 7 consensus PKC phosphorylation sites of AE3fl were individually mutated to alanine. Mutation of serine 67 of AE3 prevented the PMA-induced increase of anion transport activity. Inhibition of MEK1/2 by PD98059 (50 micromol/L) did not affect the response of AE3fl to Ang II, indicating that PKC directly phosphorylates AE3fl. We conclude that following Ang II stimulation of cells, PKCepsilon phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium.

High-dose chemotherapy with autologous stem-cell rescue in patients with recurrent and high-risk pediatric brain tumors.

PURPOSE: We treated 49 patients with recurrent or poor-prognosis CNS malignancies with high-dose chemotherapy regimens followed by autologous marrow rescue with or without peripheral-blood stem-cell augmentation to determine the toxicity of and event-free survival after these regimens. PATIENTS AND METHODS: Nineteen patients had medulloblastomas, 12 had glial tumors, seven had pineoblastomas, five had ependymomas, three had primitive neuroectodermal tumors, two had germ cell tumors, and one had fibrosarcoma. Thirty-seven received chemotherapy with cyclophosphamide 1.5 g/m2 daily x 4 and melphalan 25 to 60 mg/m2 daily x 3. Nine received busulfan 37.5 mg/m2 every 6 hours x 16 and melphalan 180 mg/m2 (n = 7) or 140 mg/m2 (n = 2). Three received carboplatin 700 mg/m2/d on days -7, -5, and -3 and etoposide 500 mg/m2/d on days -6, -4, and -2. All patients received standard supportive care. RESULTS: Eighteen of 49 patients survive event-free 22+ to 55+ months (median, 33+) after transplantation, including nine of 16 treated before recurrence and nine of 33 treated after recurrence. There was one transplant-related death from pulmonary aspergillosis. Of five patients assessable for disease response, one had a partial remission (2 months), one has had stable disease (55+ months), and three showed progression 2, 5, and 8 months after transplantation. CONCLUSION: The toxicity of these regimens was tolerable. Certain patients with high-risk CNS malignancies may benefit from such a treatment approach. Subsequent trials should attempt to determine which patients are most likely to benefit from high-dose chemotherapy with autologous stem-cell rescue.

Higher levels of mucosal antibody to pneumococcal vaccine candidate proteins are associated with reduced acute otitis media caused by Streptococcus pneumoniae in young children.

Mucosal immunity has a crucial role in controlling human respiratory tract infections. This study characterizes the naturally acquired mucosal antibody levels to three Streptococcus pneumoniae (Spn) protein antigens, pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and pneumolysin (Ply), and assesses the association of the mucosal antibody levels with occurrence of acute otitis media (AOM) caused by Spn. Both nasopharyngeal (NP) immunoglobulin G (IgG) and IgA levels to all three proteins slightly decreased in children from 6 to 9 months of age and then gradually increased through 24 months of age. Spn NP colonization was associated with higher mucosal antibody levels to all three proteins. However, children with Spn AOM had 5-8-fold lower IgG and 3-6-fold lower IgA levels to the three proteins than children without AOM but asymptomatically colonized with Spn. Antigen-specific antibody levels in the middle ear fluid (MEF) were correlated with antibody levels in the NP. Children with AOM caused by Spn had lower antibody levels in both the MEF and NP than children with AOM caused by other pathogens. These results indicate that higher naturally acquired mucosal antibody levels to PhtD, PcpA and Ply are associated with reduced AOM caused by Spn.

Penicillin failure in streptococcal tonsillopharyngitis: causes and remedies.

UNLABELLED: Penicillin administered for 10 days has been the treatment of choice for group A beta-hemolytic streptococcal tonsillopharyngitis since the 1950s. The bacteriologic failure rate of 10 days of penicillin therapy ranged from approximately 2 to 10% until the early 1970s. Beginning in the late 1970s bacteriologic and clinical failure rates with penicillin therapy began to increase steadily over time and are now reported to be approximately 30%. The primary cause of penicillin treatment failure in streptococcal tonsillopharyngitis may be lack of compliance with the 10-day therapeutic regimen. Other causes of penicillin treatment failure include reexposure to Streptococcus-infected family members or peers; copathogenicity, in which bacteria susceptible to a class of drugs are protected by other, colocalized bacterial strains that lack the same susceptibility; antibiotic-associated eradication of normal protective pharyngeal flora; and penicillin tolerance, whereby streptococcal bacteria repeatedly or continuously exposed to sublethal concentrations of antibiotic become increasingly resistant to eradication. Although 10 days of penicillin therapy is effective in the management of tonsillopharyngitis for many patients, multiple factors may, singly or together, cause treatment failure. A number of antibiotics, particularly the cephalosporins, have been demonstrated to be superior to penicillin at eradicating group A beta-hemolytic Streptococcus, and several are effective when administered for 4 to 5 days. CONCLUSIONS: Ten days of penicillin therapy may not be the best therapeutic choice for all pediatric patients. Other antibiotics, shortened courses of the cephalosporins in particular, may be preferable in some cases.

Toxicity, pharmacology and feasibility of administration of PEG-L-asparaginase as consolidation therapy in patients undergoing bone marrow transplantation for acute lymphoblastic leukemia.

We attempted to administer PEG-L-asparaginase (PEG-L-A) following hematologic recovery to 38 patients undergoing autologous or allogeneic marrow transplantation for acute lymphoblastic leukemia (ALL). Twenty-four patients (12 of 22 receiving allogeneic and 12 of 16 receiving autologous transplants) received between one and 12 doses of PEG-L-A, including nine who completed the planned 12 doses of therapy. The toxicities encountered were similar to those observed in non-transplanted patients undergoing therapy with PEG-L-A and included allergic reactions, pancreatitis, weight loss, hypoalbuminemia, and low levels of anti-thrombin III. Of the 24 who received the drug, eight remain in remission. Of 12 patients in second remission at the time of transplantation who received PEG-L-A, five of seven who received allogeneic and two of five who received autologous transplants remain in remission, 16+ to 46+ months from transplant. While PEG-L-A could be administered to most of the patients undergoing marrow transplantation for ALL, most patients either relapsed while receiving the drug or developed toxicities which resulted in abbreviated courses. At this time, we cannot recommend PEG-L-A as single agent, post-BMT chemotherapy.

Carbonic anhydrase: in the driver's seat for bicarbonate transport.

Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO(2) + H(2)O <=> HCO(3)(-) + H(+). These enzymes therefore both produce HCO(3)(-) for transport across membranes and consume HCO(3)(-) that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO(3)(-) across cells and epithelial layers. Plasma membrane anion exchange proteins (AE) transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO(3)(-) transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII) via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl(-)/HCO(3)(-) exchange activity in transfected HEK293 cells. Treatment of AE1-transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wild-type CAII from AE1, a loss of transport activity would be observed if binding to the AE1 C-terminus is required for transport.

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.

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.

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.

Acute otitis media disease management.

A first step in management decisions regarding otitis media must focus on accurate diagnosis to distinguish normal from acute otitis media (AOM) from otitis media with effusion (OME) or a retracted tympanic membrane without middle ear effusion. There are several classification schemes for AOM that may impact management decisions: patients with acute, persistent, recurrent, or chronic AOM may have a different distribution of bacterial pathogens and a different likelihood of success from antimicrobial therapy. Patient age, prior treatment history and daycare attendance are other important variables. The natural history of AOM without antibiotic treatment is generally favorable; however, from the few studies available, this is difficult to quantitate because the diagnosis was infrequently confirmed by tympanocentesis leaving the possibility that many patients entered into these trials may not have had bacterial AOM. Antibiotic choices should reflect pharmacokinetic/pharmacodynamic data and clinical trial results demonstrating effectiveness in eradication of the most likely pathogens based on tympanocentesis sampling and antibiotic sensitivity testing. Thereafter, compliance factors such as formulation, dosing schedule and duration of treatment and accessibility factors such as availability and cost should be taken into account. The increasing prevalence of antibiotic resistance among AOM pathogens and the changing susceptibility profiles of these bacteria should be considered in antibiotic selection. Current best practice recommends amoxicillin for uncomplicated AOM; continuing or switching to an alternative antibiotic based on clinical response after 48 hours of therapy; and selection of second line antibiotics as first line choices when the patient has already been on an antibiotic within the previous month or is otitis prone. Preferred second-line agents frequently noted in various guidelines include amoxicillin/clavulanate, cefdinir, cefpodoxime, cefprozil, and cefuroxime. Three injections of ceftriaxone or gatifloxacin (when approved) or diagnostic/therapeutic tympanocentisis (when approved) become a third-line treatment option. No single antibiotic or management strategy is ideal for all patients.

Anion exchangers in the red cell and beyond.

The anion exchanger genes (AE1-3) encode a family of transport proteins responsible for the electroneutral exchange of bicarbonate and chloride across membranes. These transporters are important in processes such as pH regulation and bicarbonate metabolism. This article reviews recent progress in this field based on presentations made at a satellite workshop on anion exchangers held in conjunction with the 8th Fisher Winternational Symposium on Cellular and Molecular Biology entitled "Membrane proteins in health and disease." The transmembrane topology of AE1 has been refined using various combinations of protein chemistry and site-directed mutagenesis. The use of specific inhibitors and novel expression systems continues to reveal fundamental features of the anion exchanger mechanism and its regulation. The importance of anion exchangers in blood and kidney diseases is underscored by the identification and characterization of a plethora of novel mutations in the AE1 gene. Investigations of anion exchanger structure and function have moved beyond studies of the red cell protein into the larger arenas of cellular and molecular biology.

Transport activity of AE3 chloride/bicarbonate anion-exchange proteins and their regulation by intracellular pH.

Plasma membrane Cl(-)/HCO(3)(-) anion-exchange (AE) proteins contribute to regulation of intracellular pH (pH(i)). We characterized the transport activity and regulation by pH(i) of full-length AE3 and the cardiac isoform, AE3c, both of which are expressed in the heart. AE3c is an N-terminal variant of AE3. We also characterized AE1, AE2 and a deletion construct (AE3tr) coding for the common region of AE3 and AE3c. AE proteins were expressed by transient transfection of HEK-293 cells, and transport activity was monitored by following changes of intracellular pH or intracellular chloride concentration associated with anion exchange. Transport activities, measured as proton flux (mM H(+).min(-1)), were as follows: AE1, 24; AE2, 32; full-length AE3, 9; AE3c, 4 and AE3tr, 4. The wide range of transport activities is not explained by variation of cell surface processing since approx. 30% of each isoform was expressed on the cell surface. pH(i) was clamped at a range of values from 6.0-9.0 to examine regulation of AE proteins by pH(i). Whereas AE2 was steeply inhibited by acid pH(i), AE1, AE3 and AE3c were essentially insensitive to changes of pH(i). We conclude that AE3 and AE3c can contribute to pH(i) recovery after cellular-acid loading.

Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1.

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2, 2'-disulfonate) and DNDS (4,4'-dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the regions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate binding cause anion transport inhibition, identification of the DNDS binding regions may localize the substrate binding region of the protein. Cysteine residues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label these residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS. DNDS reduced the ability to label residues in the regions around G565, S643-M663, and S731-S742. We interpret these regions either as (i) part of the DNDS binding site or (ii) distal to the binding site but undergoing a conformational change that sequesters the region from accessibility to biotin maleimide. DNDS alters the conformation of residues outside the plane of the bilayer since the S643-M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bilayer. We conclude that the TM7-10 region of AE1 is central to the stilbene disulfonate and substrate binding region of AE1.

High level expression, partial purification, and functional reconstitution of the human AE1 anion exchanger in Saccharomyces cerevisiae.

Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.

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.