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.

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.

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.

A novel mutation in the anion exchanger 1 gene is associated with familial distal renal tubular acidosis and nephrocalcinosis.

OBJECTIVE: The anion exchanger gene (AE1) or band 3 encodes a chloride-bicarbonate (Cl(-)/HCO(3)(-)) exchanger expressed in the erythrocyte and in the renal alpha-intercalated cells involved in urine acidification. The purpose of the present study was to screen for mutations in the AE1 gene in 2 brothers (10 and 15 years of age) with familial distal renal tubular acidosis (dRTA), nephrocalcinosis, and failure to thrive. METHODS: AE1 mutations were screened by single-strand conformation polymorphism, cloning, and sequencing. RESULTS: A complete form of dRTA was confirmed in the 2 affected brothers and an incomplete form in their father. All 3 were heterozygous for a novel 20-bp deletion in exon 20 of the AE1 gene. This deletion resulted in 1 mutation in codon 888 (Ala-888-->Leu) followed by a premature termination codon at position 889, truncating the protein by 23 amino acids. As band 3 deficiency might lead to spherocytic hemolytic anemia or ovalocytosis, erythrocyte abnormalities were also investigated, but no morphologic changes in erythrocyte membrane were found and the osmotic fragility test was normal. CONCLUSIONS: A novel mutation in the AE1 gene was identified in association with autosomal dominant dRTA. We suggest that RTA be considered a diagnostic possibility in all children with failure to thrive and nephrocalcinosis.