Paulo Roberto Moura Lima

Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

Band 3Tambaú: a de novo mutation in the AE1 gene associated with hereditary spherocytosis. Implications for anion exchange and insertion into the red blood cell membrane

Abstract:  Hereditary spherocytosis (HS) is attributed to red blood cell membrane protein defects, caused by mutations in ankyrin, spectrin, band 3 and protein 4.2. In this study, the presence of band 3 mutations was investigated in a patient presenting mild HS and band 3 deficiency. Using single strand conformation polymorphism analysis, a shift in exon 16 of the band 3 gene was found. DNA sequencing revealed a point mutation 2102 T>C, changing methionine at position 663 to lysine. The M663K substitution was not found in either the parents or in the siblings, and the restriction fragment length polymorphism analysis of 100 alleles from a random Brazilian population did not reveal this mutation, suggesting that this gene defect is more likely to be a de novo mutation, causing HS. Flow cytometry of eosin‐5‐isothiocyanate (EITC)‐labelled erythrocytes showed, in the patient, 54% of band 3 protein content vs. 78% based on the sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, suggesting that flow cytometry is a more sensitive method and may be used as a diagnostic tool in membrane disorders related to band 3 deficiency. The characterisation of novel AE1 mutations is helpful to improve the understanding of the role of band 3 protein in cell physiology.

α‐cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue‐specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two‐hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C‐terminal region of the α‐cardiac actin. Immunoprecipitation of whole rat heart using an anti‐actin antibody, immunoblotted with anti‐human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with α‐cardiac actin in cardiomyocyte, and we suggest that the binding occur “in situ,” in the intercalated disc, a site of cell–cell contact and attachment of the sarcomere to the plasma membrane.

Arginine 490 is a hot spot for mutation in the band 3 gene in hereditary spherocytosis.

To the Editor: Hereditary spherocytosis ( H S ) is a common inherited haemolytic anaemia characterised by the presence of red cells with a shortened life-span caused by preferential trapping in the spleen. Mutations in the AEI gene, which encode the erythrocyte protein band 3, have been associated with hereditary spherocytosis in band 3 deficient patients (1-15). These defects are heterogeneously distributed by the cytoplasmic and transmembrane domains. We studied one family and one unrelated patient presenting hereditary spherocytosis characterised by haemolytic anaemia, increased osmotic fragility of red cells and the presence of spherocytes and pincered cells in a peripheral blood smear. Densitometric scans of Coomassie blue-stained SDS-PAGE (16) showed a deficiency of band 3. The pedigree of the family studied is shown in Fig. 1. Clinical and haematological values from HS patients are summarised in Table 1. The molecular study was based on genomic DNA extraction, exon amplification by PCR, non-radioactive single-strand conformation polymorphism analysis (SSCP), and DNA sequencing of abnormal SSCP patterns. The AEl gene screening revealed, in exon 13, a new mutation at nucleotide 1583, changing the arginine 490 (CGC) to histidine (CAC) (Fig. 2). We named this mutation Band 3 Pinhal. The Arg 490 + His mutation was associated

ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

Molecular differentiation of Leishmania protozoarium using CdS quantum dots as biolabels

In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λexc=795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.

High fluorescent and stable semiconductor quantum dots for red blood cells labeling

We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.