Phytochemical and proximate analysis of ipomoea cairica tuber

The analyses for nutrients of Ipomoea cairica tuber were carried out and the phytochemical and proximate parameters were determined. The phytochemical analyses looked into the alkaloid, flavonoid, cyanogenic glycoside, tannin, phytate and saponin contents of Ipomoea cairica tuber while the proximate composition determination was aimed at the protein, carbohydrate, moisture, lipid, ash and fibre contents. The methods applied for these analytical determinations were mainly those of the Association of Official Analytical Chemist (AOAC, 2005). Each type of analysis (whether proximate or phytochemical) were carried out in triplicate and the mean values determined. The phytochemical analyses showed that Ipomoea cairica tuber had; flavonoid (1.52±0.03 %), Alkaloid (2.10±0.28 %), Tannin (0.05 %), Saponin (1.24±0.04 %), Cyanogenic glycoside (0.05 %), and phytate (0.02 %). The proximate analysis showed that Ipomoea cairica tuber had; Carbohydrate (11.7±0.1 %), protein (4.2±0.2 %), ash (0.6±0.1 %), lipid (0.51±0.07 %), moisture (72.7±0.2 %), and fibre (10.3±0.6 %). These results show that Ipomoea cairica tuber can be used as a substitute for other mostly consumed carbohydrate foods like yam and cocoyam.

Age – Related changes in serum biochemical parameters in the domestic rabbit (Oryctolagus cuniculus)

The influence of age on serum biochemical components in the domestic rabbit was investigated. The serum total protein (g/dL), albumin (g/dL), globulin (g/dL), creatinine (mg/dL), urea mg/dL, cholesterol (mg/dL), Alkaline phosphatase (ALP; iu/l), Aspartate aminotranferase (AST; iu/l) and Alanine amimotransferase (ALT; iu/l) were assessed in 24 male rabbits at 7, 14 and 21 weeks of age to represent weaners, growers and pubertal stages. Data were analysed using descriptive statistics and analysis of variance at á . Results of 0.05 serum biochemistry revealed a steady significant increase in total protein (4.2±0.3; 5.3±0.5; 6.9±0.2), albumin (2.2±0.7; 2.8±0.4; 4.0±0.2), globulin (1.9±0.3; 2.5±0.4, 2.9±0.2), cholesterol (49.5±1.1; 56.4±4.2; 62.4±5.4) and creatinine (0.8±0.1; 1.2±0.1; 1.9±0.3) at 7, 14 and 21 weeks respectively. Serum urea levels were similar across the ages. The values recorded for ALP and AST at 7 weeks (39.6±7.5; 101.1±6.7) and 14 weeks (38.7±6.4; 104.5±8.7) were significantly higher than the 21 weeks (33.5±2.7; 95.8±5.9) while ALT at 7 weeks (27.33±5.6) was significantly lower than 14 (38.2±3.9) and 21 weeks (37.4±3.0). The age of the rabbits had a significant effect on their serum biochemical parameters. They should therefore be grouped according to age when setting serum reference values for them.

The Cytoskeletal Binding Domain of Band 3 Is Required for Multiprotein Complex Formation and Retention during Erythropoiesis

The bicarbonate/chloride exchanger protein band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex required for vertical association between the plasma membrane and the underlying spectrin cytoskeleton. A wealth of knowledge, derived from a host of varied studies including in vitro binding assays, work on mature erythrocytes and in other cellular systems have identified a number of binding partners including ankyrin, adducin and protein 4.2 amongst others. However, studies of the role that band 3 and the establishment of its connectivity with the cytoskeleton play both in assembly of multiprotein complexes during erythropoiesis and in particular in protein retention during enucleation have been understandably limited by the technical challenges associated with study of this protein within its unique native cellular context. The complete absence of band 3 in human erythrocytes has only been reported once, in a Portuguese patient with severe hereditary spherocytosis and distal renal tubular acidosis resulting from homozygosity for a V488M band 3 mutation (band 3 Coimbra). In this study, we used in vitro culture of erythroblasts derived from this patient as well as shRNA mediated depletion of band 3 to investigate the development of a band 3 deficient erythrocyte membrane and to specifically assess the formation, stability and retention of band 3 dependent protein complexes in the absence of this core protein during erythropoiesis and erythroblast enucleation. We demonstrate that the mutant band 3 Coimbra protein is expressed at very low but detectable levels during erythropoiesis but does not reach the cell surface and is not rescued by interaction with wild type protein. Failure to traffic to the plasma membrane and rapid degradation during erythropoiesis accounts for the absence of band 3 in Coimbra erythrocytes. The absence of plasma membrane expression of band 3 results in secondary deficiencies of a host of band 3 associated membrane proteins that we quantitatively show result predominantly from reduced plasma membrane expression during erythropoiesis compounded by impaired retention in the nascent reticulocyte membrane during erythroblast enucleation. In order to explore the importance of the capacity of band 3 to associate with the cytoskeleton for surface expression of this protein and its associated multiprotein complex binding proteins, immature band 3 Coimbra patient erythroblasts were lentivirally transduced with N terminally GFP-tagged wild type band 3 or band 3 mutants with absent or impaired ability to associate with the cytoskeleton. We demonstrate for the first time the ability to restore expression of band 3 to normal levels in this uniquely compromised patient and to rescue key secondary protein deficiencies arising from the absence of band 3 in reticulocytes. Exogenous expression levels of band 3, monitored by GFP intensity, correlate directly with degree of rescue of a variety of band 3 associated proteins. When expressed in band 3 deficient Coimbra erythroblasts, the band 3 membrane domain, which is unable to associate with the cytoskeleton, exhibits an increased partitioning to the plasma membrane surrounding the extruded nuclei compared to wild type band 3 and fails to rescue reticulocyte membrane retention of band 3 associated proteins. Expression of the kidney isoform of band 3, which is unable to bind ankyrin but retains the binding site for the cytoskeletal accessory protein, protein 4.2 results in partial rescue of the protein 4.2 dependent CD47 only. This demonstrates the importance of band 3 association with the cytoskeleton for efficient retention of band 3 associated proteins during erythroblast enucleation. Interestingly, whilst both exhibit reduced reticulocyte membrane retention relative to wild type, a significant proportion of both band 3 membrane domain and kidney band 3 is retained in the reticulocyte membrane following erythroblast enucleation indicating that cytoskeletal attachment of band 3 is not the sole determinant of partitioning during this complex process. This study advances our understanding of the mechanisms by which the properties of band 3 influence the sculpting and composition of the erythrocyte membrane and highlights the role of this protein as a core for assembly and stabilisation of key membrane proteins in both the early and late stages of terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

CD47 Is Dependent on the Actin Cytoskeleton for Its Membrane Stability Prior to Protein 4.2 Expression during Early Erythroblast Differentiation

CD47 is a ubiquitously expressed ‘Marker of Self’ that protects cells from phagocytosis, through recognition by SIRPα on macrophages (Oldenborg et al Science 2000). CD47 was originally isolated on ovarian tumour cells (Poels et al J Natl Cancer Inst 1986) and has subsequently been detected on leukemic stem cells, where increased CD47 levels ensure immune evasion (Jaiswal et al Cell 2009). CD47 is also a ‘Marker of Self’ on red cells, but is reduced at the cell surface in certain patients with Hereditary Spherocytosis. In red cells, ~60% of CD47 is connected to the cytoskeleton (Dahl et al Blood 2004). Cytoskeletal connectivity of CD47 in the red cell membrane is dependent on the band 3 complex associated protein 4.2, demonstrated by an ~80% reduction in CD47 levels in protein 4.2 null red cells (Mouro-Chanteloup et al Blood 2003). Previous work (van den Akker et al Haematologica 2009) established that CD47 becomes dependent on protein 4.2 at the basophilic erythroblast stage (48 hours post-differentiation), but it is unknown what interactions support CD47 membrane stability prior to protein 4.2 expression during expansion and early erythroid differentiation. CD47 mRNA is alternatively spliced giving rise to four potential isoforms. The most abundant isoforms are form 2, expressed in all bone-marrow derived cells, and form 4 (and form 3), found predominantly in neural tissues (Reinhold et al J Cell Sci 1995). CD47 isoform 2 is the only form expressed on mature red cells, but we hypothesized that expression of other CD47 isoforms with different trafficking or binding characteristics could explain the independence of CD47 prior to band 3 complex assembly. Using specific polyclonal antibodies to multiple CD47 isoforms, we demonstrate that isoform 2 is expressed prior to and throughout in vitroerythroid differentiation. CD47 isoforms 3 and 4 were detected by western blotting until the late polychromatic erythroblast stage (96 hours post-differentiation), but only CD47 isoform 2 was detected at the cell surface. Therefore, we next hypothesised that CD47 must interact with another protein or exhibit different trafficking characteristics to maintain its membrane stability early during terminal differentiation. To identify a candidate protein or associated protein complex, CD47 was immunoprecipitated from expanding erythroblasts (Exp), proerythroblasts (T0), and basophilic erythroblasts (T48), and analysed via Nano-LC mass spectroscopy. In Exp and T0 erythroblasts, CD47 pulled down actin and multiple actin-associated proteins. These interactions were not observed in T48 erythroblasts, corresponding to the time during terminal differentiation when CD47 is dependent on protein 4.2. To confirm a dependence on actin for CD47 membrane stability, well-characterised drugs that disrupt actin dynamics were employed. CD47 expression at the cell membrane, as judged by flow cytometry, was markedly reduced within 30 minutes using actin stabilising drugs (Cytochalasin D (5µM): Exp 13.7±5.4% versus T48 0.5±5.7%; Latrunculin A (1µM): Exp 18.9±3.5% versus T48 9.9±5.9%, of the DMSO control), and destabilising drug (Jasplakinolide (1µM): Exp 24.2±1.9% versus T48 -6±1.8%, of the DMSO control), until the basophilic erythroblast stage. In K562 cells, which predominantly express CD47 isoforms 3 and 4, a larger actin dependency is observed (37±14.9% reduction in CD47 with Cytochalasin D versus a DMSO control) suggesting that dependence on actin by CD47 is not isoform specific. In summary, we propose a role for actin in the maintenance of CD47 at the cell surface before and during early erythroid differentiation. We have shown that CD47 isoform 2 is the major isoform present at the cell surface and that this version is initially dependent on the actin cytoskeleton for its membrane stability by an as yet undetermined mechanism. Once band 3 complex assembly initiates at the surface of the basophilic erythroblast (48 hours post-differentiation), CD47 is selectively incorporated via an interaction with protein 4.2, and is preferentially retained whilst the actin cytoskeleton remodels. In addition to explaining how CD47 expression is maintained during the formation of the red cell membrane, this work raises the possibility that the dependence on actin by CD47 for its membrane stability in hematopoietic stem cells may be exploited for the development of therapeutics that render the leukemic cells susceptible to phagocytosis. Disclosures No relevant conflicts of interest to declare.

Protein 4.2 interaction with hereditary spherocytosis mutants of the cytoplasmic domain of human anion exchanger 1

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.

Mapping the Assembly of Band 3 and Rhesus Multi-Protein Complexes During Erythropoiesis

Abstract 812 Band 3 forms the core of a large multiprotein complex in the erythrocyte membrane, the Band 3 macrocomplex, which also includes proteins of the Rhesus complex (Rh and RhAG). Mutations in genes encoding proteins within this complex can result in hereditary spherocytosis with varying severity. The effect of distinct mutations and deficiencies in proteins of the Band 3 macrocomplex has been studied in detail in mature erythrocytes. This revealed important functional and structural properties of individual proteins and their relationships with other proteins within the Band 3 macrocomplex. Nevertheless, considerably less is know about the spatio-temporal mechanisms that direct the formation of the Band 3 macrocomplex, and that may explain the aberrations in the complex observed in spherocytosis. Therefore, we studied expression and mutual interactions of proteins of the band3 macrocomplex during development of proerythroblasts to reticulocytes. Using confocal microscopy and western blotting, significant pools of intracellular Band 3 and RhAG were found in the basophilic normoblast. These intracellular pools gradually decreased in the polychromatic normoblast and were absent or low in the orthochromatic normoblast and reticulocytes, while surface expression increased. We used pronase treatment of intact cells to remove extracellular epitopes of BRIC 6 (Band 3 antibody) and LA1818 (RhAG antibody) to study the mechanism by which the intracellular pool of Band 3 and RhAG contributes to formation of the Band 3 complex on the cell surface. Pronase treatment of cells incubated with cycloheximide to block protein synthesis resulted in a reduced but still significant reappearance of BRIC6 (Band 3) and LA1818 (RhAG) epitopes on the plasma membrane confirming the presence of intracellular Band 3 and RhAG pools. It also showed that the bulk of Band 3 and RhAG is synthesized and trafficked to the membrane between the early basophilic and polychromatic stage. Immuneprecipitation of Band 3 from cell lysates of pronase treated cells pre-treated with brefeldin A to collapse the Golgi showed no increase in co-immuneprecipitated protein 4.2 albeit an increase in intracellular Band 3 expression. This suggests that protein 4.2 and Band 3 interact in the first Golgi compartment or late ER. In addition, pre-treatment of cells with cycloheximide prior to pronase treatment resulted in depletion of the intracellular Band 3 and co-immuneprecipitated protein 4.2 pool indicating that Band 3 and protein 4.2 traffic as a complex to the plasma-membrane. We were unable to co-immuneprecipitate Rh or Band 3 with intracellular pools of RhAG, whereas Rh was co-immuneprecipitated with RhAG from the plasma-membrane and from total cell lysates. Knockdown of RhAG in differentiating erythroblasts revealed a concomitant drop in membrane expression of Rh, leaving Band 3 unaffected, indicating that plasma-membrane expression of Rh but not Band 3 is dependent on RhAG. In conclusion, despite the described association between the RhAG complex and the Band 3 complex in erythrocytes, the data suggest that the Band 3-protein 4.2 complex traffics and assembles independently from Rh and RhAG during erythroid differentiation. The experiments suggest that Rh and RhAG do not traffic as a complex to the plasma-membrane but probably assemble in the plasma-membrane. The RhAG knockdown experiments suggest that the dependency of Rh on RhAG as observed in Rhnull syndrome erythrocytes (“Rh regulator type”) originates early during erythropoiesis. Band3 surface expression was not affected upon RhAG knock down, which re-produced the unperturbed Band 3 levels seen in these patients. Disclosures: No relevant conflicts of interest to declare.

Disorders of the red cell membrane

The integrity of the red-cell membrane depends on molecular interactions between proteins and protein–lipid interactions: vertical interactions stabilize the membrane lipid bilayer; horizontal interactions provide resistance against shear stress. This disorder affects 1 in 25 000 individuals of northern European descent. There is typically a dominant family history, but the condition is genetically heterogeneous: combined spectrin and ankyrin deficiency is the most common defect observed, followed by band 3 deficiency, isolated spectrin deficiency, and protein 4.2 deficiency. These affect vertical membrane interactions with loss of surface area relative to red-cell volume....