Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls.
Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates cellular properties performed by these and/or related proteins. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 reversibly with high affinity, these observations raise the hypothesis that hemoglobin might regulate erythrocyte properties through its reversible, oxygenationdependent association with band 3. To explore this hypothesis, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that (1) deoxyHb binds to residues 12-23 of band 3; (2) mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction would have been possible had it been beneficial for survival; (3) Hb does not bind to 2 other sequences in band 3 despite their high sequence homology to residues 12-23, and (4) IntroductionSeveral lines of evidence suggest that the oxygenation state of the erythrocyte (RBC) regulates multiple erythrocyte properties. First, the activities of many membrane solute transporters change with the oxygen content of the cell, including volume regulatory transporters (eg, Na ϩ /H ϩ exchanger), cation coupled Cl Ϫ cotransporters, amino acid transporters, and ion channels. [1][2][3][4][5][6][7][8][9][10] The K/Cl cotransporter, for example, is reported to be 20-fold more active in oxygenated than in deoxygenated RBCs. 9,10 Second, erythrocyte metabolism is modulated by the O 2 tension of the medium. Thus, glucose flux through the pentose phosphate pathway proceeds twice as rapidly in oxygenated as in deoxygenated cells, and glucose consumption in glycolysis is inhibited by oxygenation. 11 Third, erythrocyte deoxygenation may affect membrane structural properties. For example, deoxygenated hemoglobin (Hb) (but not oxygenated Hb [oxyHb]) competes with ankyrin for binding to band 3, and band 3 retention in detergent-extracted membrane skeletons is greatly reduced in skeletal pellets from deoxygenated cells (M. Stefanovic and P.S.L., unpublished data, December 2005). Furthermore, ankyrin epitopes are more accessible to antiankyrin antibodies in deoxygenated than in oxygenated RBCs. Taken together, these latter findings imply a looser association of ankyrin with the membrane in deoxygenated than in oxygenated erythrocytes.In searching for a mechanism to account for oxygenation effects on red cell properties, identification of both the O 2 sensor and downstream effector was considered important. A major candidate for the O 2 sensor was presumed to be Hb, because the O 2 dependence of the above properties generally follows the O 2 dissociation curve of Hb. 4,12,13 The downstream effector has been more uncertain, but band 3 has represented the prime candidate, because it constitutes the only established binding site of Hb on the membrane. Moreover, multiple observation...
The determinants of sickle red blood cell (RBC) life span have not been welldefined but may include both intrinsic factors (eg, the tendency to sickle) and extrinsic factors (eg, the capacity of the reticuloendothelial system to remove defective RBCs).
SP-A is an abundant pulmonary surfactant-associated protein whose expression is controlled in a cell- and developmental-specific manner. To analyze regulation of SP-A gene expression, the murine SP-A gene was cloned and sequenced. The murine DBA/2J gene was approximately 4.6 kb in length comprised of six exons and five introns. Three mRNAs of 3.0, 1.7, and 0.9 kb were detected by Northern blot analysis of murine lung mRNA. Expression of the SP-A mRNAs was first detected at day 15 of gestation and increased dramatically before birth. A single SP-A gene was detected in the DBA/2J mouse genome. SP-A mRNA was detected in lung but not in the gastrointestinal tract, kidney, brain, liver, or heart and was detected by in situ hybridization in bronchial and alveolar cells of the murine lung. Primer extension analysis with a primer to exon three revealed two extension products differing by 9 bp in length, suggesting two closely juxtaposed transcription initiation sites. Chimeric gene(s) containing 1.8 kb of 5' SP-A sequences and the bacterial chloramphenicol acetyltransferase gene were expressed in pulmonary adenocarcinoma cells and in HeLa cells. Expression of the murine SP-A gene is partially controlled by non-cell-selective transcriptionally active sequences.
Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.
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