Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.     

Immunohistochemical localization of membrane and alpha-granule proteins in human megakaryocytes: application to plastic-embedded bone marrow biopsy specimens

Using a new technique for antigen localization, we have demonstrated platelet proteins in megakaryocytes in plastic-embedded biopsy specimens of normal human bone marrow. In a series of 25 specimens, megakaryocytes showed labeling with antibodies to the integral membrane glycoproteins IIIa, IIb, and the IIb-IIIa complex; granule membrane protein 140; and five alpha-granule matrix proteins: thrombospondin, factor VIII-related antigen, beta-thromboglobulin, platelet factor 4, and fibrinogen. The antibodies to the membrane glycoproteins IIIa, IIb, and IIb-IIIa produced diffuse cytoplasmic staining and heavier staining on the plasma membrane, whereas the antibodies to the alpha-granule matrix proteins produced a distinct granular staining within the cytoplasm. Staining for granule membrane protein 140 was also granular in distribution. Rare mononuclear cells consistent with megakaryocyte precursors were labeled with these markers. Other enzyme histochemical and lectin-binding studies showed that the enzyme alpha-naphthyl acetate esterase, the lectin Ulex europaeus I, and the periodic-acid Schiff reaction were consistent, but not specific, markers of megakaryocytes. This immunohistochemical technique should facilitate the examination of qualitative and quantitative changes in megakaryocytes in a variety of physiologic and pathologic processes.     

Influence of ATP-Binding Cassette Polymorphisms on Neurological Outcome After Traumatic Brain Injury

Background

As important mediators of solute transport at the blood–brain and blood–cerebrospinal fluid barriers, ATP-binding cassette (ABC) transporters (including ABCB1, ABCC1, and ABCC2), impact the bioavailability of drugs and endogenous substrates in the brain. While several ABCB1, ABCC1, and ABCC2 single nucleotide polymorphisms (SNPs) have been identified, their impact on outcome after traumatic brain injury (TBI) is unknown.

Hypothesis

ABCB1, ABCC1, and ABCC2 SNPs are associated with Glasgow Outcome Scale (GOS) score after TBI.

Methods

DNA samples from 305 adult patients with severe TBI (Glasgow Coma Scale, GCS score ≤ 8) were genotyped for tagging SNPs of ABCB1 (rs1045642; rs1128503), ABCC1 (rs212093; rs35621; rs4148382), and ABCC2 (rs2273697). For each SNP, patients were dichotomized based on presence of variant allele for multivariate analysis to determine associations with GOS assigned at 6 months adjusting for GCS, Injury Severity score, age, and patient sex.

Results

For ABCB1 rs1045642, patients homozygous for the T allele were less likely to be assigned poor outcome versus those possessing the C allele [CT/CC; odds of unfavorable GOS = 0.71(0.55–0.92)]. For ABCC1 rs4148382, patients homozygous for the G allele were less likely to be assigned poor outcome versus those possessing the A allele [AG/AA; odds of unfavorable GOS = 0.73(0.55–0.98)].

Conclusions

In this single-center study, patients homozygous for the T allele of ABCB1 rs1045642 or the G allele of ABCC1 rs4148382 were found to have better outcome after severe TBI. Further study is necessary to replicate these very preliminary findings and to determine whether these associations are due to central nervous system bioavailability of ABC transporter drug substrates commonly used in the management of TBI, brain efflux of endogenous solutes, or both.     

Muscle architecture adaptations to knee extensor eccentric training: Rectus femoris vs. vastus lateralis

Introduction: Changes in muscle architecture induced by eccentric knee extensor training remain unclear, as well the adaptive responses of synergistic knee extensor muscles with different geometrical designs. Methods: Ultrasonography images were taken from rectus femoris (RF) and vastus lateralis (VL) of 20 male volunteers before and after a non‐training control period of 4 weeks, and additional evaluations were performed after 4, 8, and 12 weeks of isokinetic eccentric training. Results: RF and VL had significant changes in muscle architecture within the first 4 training weeks, and the adaptive response throughout the intervention was similar. Muscle thickness increased by around 7–10%, fascicle length increased 17–19%, and pennation angle was unchanged. Conclusions: Increased muscle thickness due to eccentric training was related to increased fascicle length and not to pennation angle changes. Although RF and VL have a different fascicular geometry, they had similar morphological adaptations to eccentric training. Muscle Nerve 48 : 498–506, 2013     

Advanced Pharmacy Practice Experiences in Pharmacogenomics Offered by US Pharmacy Programs

Objective. To characterize advanced pharmacy practice experiences (APPEs) with a primary focus in pharmacogenomics at schools and colleges of pharmacy in the United States.Methods. This was a cross-sectional, multicenter, observational study of pharmacogenomics APPEs at US pharmacy schools. Directors of experiential education at 146 accredited schools of pharmacy were contacted by phone and asked if their school offered a pharmacogenomics APPE. The preceptors of pharmacogenomics APPEs identified by this phone screen were sent an email with a link to an online survey that asked about their APPE offerings.Results. Of the 142 schools of pharmacy that were successfully reached via phone, 40 (28%) offered an APPE with a primary focus in pharmacogenomics. Thirty unique APPEs with pharmacogenomics as a primary focus were identified. The total number of preceptors involved in the pharmacogenomics APPEs was 33: 19 (58%) faculty preceptors and 14 (42%) non-faculty preceptors. Twenty-three of the 30 pharmacogenomics APPEs completed the survey (77% response rate). The APPE sites were diverse and included academic medical centers, community health systems, pharmacogenomic testing laboratories, and schools of pharmacy. Each pharmacogenomics APPE accommodated an average of six students per year. The APPE activities varied across sites.Conclusion. Only a small number of US pharmacy schools offer an APPE with a primary focus in pharmacogenomics. These rotations are diverse in scope and precepted by faculty or non-faculty pharmacists. The Academy should pursue opportunities to increase experiential education in pharmacogenomics.     

Colonic delivery of dexamethasone from a prodrug accelerates healing of colitis in rats without adrenal suppression

Dexamethasone-β-d-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohn's colitis. The aim of this study was to evaluate colonic delivery and efficacy of this prodrug in the rat. Distribution of dexamethasone in luminal contents and tissues of the gastrointestinal tract and in plasma was measured after oral administration of dexamethasone-β-d-glucuronide or free dexamethasone. Efficacy of the prodrug and free drug was tested in an acetic acid-induced rat colitis model. Healing of induced colitis was assessed by measuring net intestinal fluid absorption, colonic surface area of ulceration, histology, and myeloperoxidase activity. Glucocorticosteroid toxicity was evaluated with serum corticosterone and plasma adrenocorticotropic hormone levels. The drug delivery index (a measure of relative targeting efficiency) was 6.7 and 8.6 in the cecal and colonic mucosa, respectively. The prodrug was significantly more potent than free drug in improving net colonic fluid absorption while significantly reducing surface area of ulceration and histological grade in colitic rats. Treatment with free dexamethasone significantly reduced serum corticosterone levels to subnormal levels, and treatment with the prodrug maintained serum corticosterone and plasma adrenocorticotropic hormone levels near control levels. The prodrug dexamethasone-β-d-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.     

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.