IntroductionProtein 4.2 is a 72-kDa component of the red blood cell (RBC) membrane skeleton (200,000 copies per RBC) (1). An intact membrane skeleton attached to the lipid bilayer by vertical interactions between spectrin, ankyrin, protein 4.2, and band 3 (anion exchanger 1 [AE1]) is required for normal RBC membrane integrity. Defects in all of these components have been identified in hereditary spherocytosis (HS) (2). In humans, protein 4.2 defects are most common in people of Japanese descent and give rise to pleiotropic RBC morphological changes and hemolytic anemias of varying severity (3).In RBCs, protein 4.2 binds to the cytoplasmic domain of band 3 (4). In solution, protein 4.2 binds directly to spectrin, ankyrin, and protein 4.1, and promotes binding of spectrin to ankyrin-depleted inside-out vesicles (4, 5). The significance of these interactions in vivo is unknown. However, mice totally deficient in band 3 are also completely protein 4.2 deficient, suggesting that band 3 is critical for stable incorporation of protein 4.2 into the membrane skeleton (6).Hereditary spherocytes are characterized by increased Na + and decreased K + content compared with normal RBCs (2). Studies in mice and humans suggest that the cation alterations are the result of increased passive RBC membrane permeability and that the increased passive flux reflects the overall loss of membrane skeleton integrity, not the specific protein defect (7,8). Protein 4.2 has been proposed to negatively regulate band 3-mediated RBC anion transport (9), but its role in regulating RBC cation transport and intracellular cation content is unknown.We targeted the erythrocyte protein 4.2 gene (Epb4.2) in embryonic stem (ES) cells to create a null mutation (4.2 -/-) in mice. Deficiency of protein 4.2 in mice results in mild HS. Normal amounts of spectrin and ankyrin are assembled onto the membrane, and the membrane skeleton architecture is intact. The band 3 content of the membrane is decreased. Contrary to previous reports, band 3-mediated anion transport in not increased in 4.2 -/-RBCs, but is decreased. Protein 4.2-deficient RBCs show significant changes in RBC cation transporter activities. These changes are in marked contrast to those obtained in spectrin-and ankyrin-deficient mouse and human red cells (7,8), suggesting that specific membrane skeleton defects differentially influence cation transporter activities. Our results show that protein 4.2 is important in the maintenance of normal surface area in RBCs and is required for normal RBC cation transport. resulting in dehydration. The passive Na + permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4.2 -/-RBCs are significantly increased. Protein 4.2 -/-RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2 -/-RBCs compared with controls. The increased passive Na + permeability of 4.2 -/-RBCs is also dependent on c...

Viral shedding of HSV occurs frequently in infected individuals. HSV is shed asymptomatically from multiple anatomical sites and shedding, like exposure, is a significant risk for transmission. However, the relationship between shedding frequency, viral titer and transmission is unknown. HSV-2 shedding is affected by the site and time since acquisition of infection. The advent of sensitive PCR techniques has shown that the magnitude and frequency of viral shedding is higher than shown previously with viral culture techniques. It has also clearly demonstrated that suppressive (daily) antiviral therapy reduces clinical and subclinical reactivation rates, and has been successfully used in the prevention of recurrent oral and genital HSV infections. A recent study has demonstrated that daily antiviral therapy with valaciclovir can significantly reduce transmission of HSV-2 between discordant heterosexual couples in monogamous relationships.

We studied the exchange of cholesterol between radioactively labeled plasma and human erythrocytes.Results from experiments in which [3H]cholesterol and ['4CJ cholesterol were exchanged sequentially into the cells and back out into unlabeled plasma, showed that transmembrane movement of cholesterol occurred with a half-time that was either less than 50 min or greater than 10 days. To obtain further information about the transmembrane movement of cholesterol, we used a technique [Jaeobson, B. S. & Branton, D. (1977) Science 195, 302-304] for exposure of the cytoplasmic surface of erythrocyte membranes. This method involved the ionic attachment of erythrocytes to derivatized glass beads followed by disruption of the cells, leaving the beads covered by membrane with the cytoplasmic surface exposed. [3HlCholesterol was exchanged into intact erythrocytes which then were attached to beads. The beads with attached membrane were incubated with phospholipid-cholesterol vesicles and the exchange of cholesterol between the membrane cytoplasmic surface and vesicles was studied. We found that [3Hlcholesterol was present at the cytoplasmic surface, indicating that transmembrane movement of cholesterol had occurred within the 2.5 hr required to complete the experiment. This result suggests that the more rapid rate of transmembrane cholesterol movement, inferred from-the experiments described above, is the one that applies.Cholesterol from erythrocyte membranes exchanges rapidly with the unesterified cholesterol of plasma lipoproteins both in vivo (1) and in vitro (2). The mechanisms involved in this exchange process are not understood. Some studies have indicated that all of erythrocyte cholesterol is exchangeable (3, 4), whereas others have shown that a significant portion does not exchange (5,6). The extent to which red cell cholesterol is available for exchange has important implications with regard both to membrane structure and to mechanisms of cholesterol exchange.It is widely believed that the lipids of the erythrocyte membrane are arranged in a bilayer that has transmembrane compositional asymmetry in its phospholipid components. The location of cholesterol in the membrane is not known and it is possible that it too is asymmetrically distributed across the bilayer. A recent study of the cholesterol content of the inner and outer halves of the erythrocyte membrane has indicated that there is cholesterol in both membrane halves, with possibly more on the outer half of the membrane (7).If cholesterol located on the cytoplasmic side of the membrane is to exchange with plasma lipoproteins, it first must move across the bilayer. Translocation across the erythrocyte membrane occurs for phosphatidylcholine. Bloj and Zilversmit (8) found a half-time for the process of 2.3 hr in rat erythrocyte ghosts; Renooij et al. (9) measured a half-time of 4.5 hr in intact rat red cells.The transbilayer movement of cholesterol in vesicles occurs with a half-time in excess of 6 days (10). In [14C]cholesterol were sequential...

Our results provide new evidence that mindfulness in youth relates to functional neural dynamics and interactions between neurocognitive networks, over time.

HeLa cell plasma membranes have been purified after binding cells to polylysinecoated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25~Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 12SI-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (-2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

No abstract

No abstract

The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector Agtll. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2+-dependent crosslinking proteins, guinea pig liver transglutaminase (proteinglutamine y-glutamyltransferase; protein-glutamine:amine vglutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular y-glutamyle-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit offactor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly, within the five contiguous consensus residues of the transglutaminase active site, Gly-Gln-Cys-Trp-Val, band 4.2 has an alanine substituted for cysteine (which is apparently essential for activity). Consistent with this active site substitution, erythrocyte membranes or inside-out vesicles, which contain band 4.2, show no evidence of transglutaminase activity by two types of in vitro assay.Band 4.2 is a major protein (5% by weight) of the human erythrocyte membrane (1). This protein has an apparent mass of 72 kDa on SDS gels but forms oligomers both in solution and on the erythrocyte membrane (2, 3). Previous studies from our laboratory have shown that band 4.2 binds to the 43-kDa cytoplasmic domain of band 3, the erythrocyte anion transport protein (3,4). While this binding is likely responsible for the association of band 4.2 with the cytoplasmic surface of the membrane, band 4.2 also has other, lessunderstood associations. We have shown that band 4.2 can bind to purified erythrocyte ankyrin in solution with a Kd -10-7 M and may also associate with band 4.1 and spectrin (4). Binding measurements and competition studies suggest that ankyrin and band 4.2 bind to distinct sites on the cytoplasmic domain of band 3 (4). While we and others have not found any effect of band 4.2 on ankyrin association with erythrocyte membranes (4-6), other studies suggest that band 4.2 may stabilize ankyrin-band 3 associations (7).While the exact function of band 4.2 on the erythrocyte membrane is not known, individuals whose erythrocytes lack or are deficient in band 4.2 have hemolytic anemia associated with spherocytic or elliptocytic erythrocytes (7,8). Absence of band 4.2 associated with spur or target erythrocytes has also been reported (9). These findings suggest that band 4.2 probably plays an important role ...