Protein deficiency balance as a predictor of clinical outcome in hereditary spherocytosis

Vertical and horizontal interactions between membrane constituents account for integrity, strength and deformability of the erythrocyte. Disruption of vertical interactions caused by membrane protein deficiencies in hereditary spherocytosis (HS), favor membrane vesiculation with development of spherocytic cells. Our aim was to evaluate the hematological and clinical presentation of HS according to the type and amount of protein deficiency. We studied 81 Portuguese individuals, 71 belonging to 21 families plus 10 unrelated subjects, and found that 51 of them were HS patients. Patients were classified as presenting mild, typical or severe HS, according to laboratory results and clinical follow-up. We performed screening tests and the standardized electrophoretic membrane protein analysis to identify and quantify protein deficiencies. We found band 3 and ankyrin deficiencies as the major causes for HS. The ratios between the value of the primary and/or secondary protein deficiencies showed significantly different values according to the severity of HS, and a significant inverse correlation with the severity of HS was observed. In mild HS, the ratios between protein deficiencies reflected equivalent protein deficiencies, while an unbalance was observed in typical HS, which was enhanced in severe HS. Our data suggest that the relative quantification of each major membrane protein and of the ratios between the values of protein deficiencies may be helpful in providing additional data about the clinical outcome of HS

Anion Exchanger 1 (Band 3) Is Required to Prevent Erythrocyte Membrane Surface Loss but Not to Form the Membrane Skeleton

AbstractThe red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1–lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis

Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax

Use of an innovative model to evaluate mobility in seniors with lower-limb amputations of vascular origin: a pilot study

<p>Abstract</p> <p>Background</p> <p>The mobility of older individuals has often been only partially assessed, without considering all important aspects such as potential (available) versus effective (used) mobilities and the physical and psychosocial factors that modulate them. This study proposes a new model for evaluating mobility that considers all important aspects, applied here to lower-limb amputees with vascular origin. This model integrates the concepts of potential mobility (e.g. balance, speed of movement), effective mobility (e.g. life habits, movements in living areas) and factors that modulate these two types of mobility (e.g. strength, sensitivity, social support, depression). The main objective was to characterize potential and effective mobility as well as mobility modulators in a small sample of people with lower-limb amputations of vascular origin with different characteristics. The second objective of this pilot study was to assess the feasibility of measuring all variables in the model in a residential context.</p> <p>Methods</p> <p>An observational and transversal design was used with a heterogeneous sample of 10 participants with a lower-limb amputation of vascular origin, aged 51 to 83, assessed between eight and 18 months after discharge from an acute care hospital. A questionnaire of participant characteristics and 16 reliable and valid measurements were used.</p> <p>Results</p> <p>The results show that the potential mobility indicators do not accurately predict effective mobility, i.e., participants who perform well on traditional measures done in the laboratory or clinic are not always those who perform well in the real world. The model generated 4 different profiles (categories) of participants ranging from reduced to excellent potential mobility and low to excellent effective mobility, and characterized the modulating factors. The evaluations were acceptable in terms of the time taken (three hours) and the overall measurements, with a few exceptions, which were modified to optimize the data collected and the classification of the participants. For the population assessed, the results showed that some of the negative modulators (particularly living alone, no rehabilitation, pain, limited social support, poor muscle strength) played an important role in reducing effective mobility.</p> <p>Conclusion</p> <p>The first use of the model revealed interesting data that add to our understanding of important aspects linked to potential and effective mobility as well as modulators. The feasibility of measuring all variables in the model in a residential context was demonstrated. A study with a large number of participants is now warranted to rigorously characterize mobility levels of lower-limb amputees with vascular origin.</p

Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion.

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis