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Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the “Dombrock” blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5′-diphosphate (ADP)–ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Doa versus Dob antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the “Dombrock” blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5′-diphosphate (ADP)–ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Doa versus Dob antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

A complete understanding of human erythropoiesis will require a robust description of transcriptional activity in hematopoietic cells that proliferate and differentiate in response to erythropoietin (EPO). For this purpose, we cultured peripheral blood mononuclear cells in the presence or in the absence of EPO and examined the transcriptional profile of those cells arising only in response to EPO. A distinct population of CD71؉؉؉؉ cells that demonstrated an average of six additional doublings in suspension culture and erythroid colony formation in methylcellulose was isolated. Suppression subtractive hybridization of mRNA isolated from those cells permitted the identification of transcribed genes. A summary of 719 expressed sequence tags (ESTs) describing 505 independent transcripts is provided here with a full analysis of each EST available at http://hembase.niddk.nih.gov. Several transcripts that matched genes previously reported in the context of erythroid differentiation including 4 cell surface proteins were expressed at this developmental stage. Active chromatin remodeling was suggested by the identification of 4 histone proteins, 4 high-mobility group proteins, 13 transcription factors, and 6 genes involved in DNA recombination and repair. Numerous genes associated with leukemic translocations were also recognized including topoisomerases I and II, nucleophosmin, Translin, EGR1, dek, pim-1, TFG, and MLL. In addition to known transcripts, 44 novel EST were discovered. This transcriptional profile provides the first genomic-scale description of gene activity in erythroid progenitor cells.

A cytokine-screening assay of cultured peripheral blood cells obtained using immune rosetting and separation of progenitors was developed to identify determinants of fetal hemoglobin (HbF) modulation during adult erythropoiesis. Among the 12 erythroid growth-promoting cytokines tested, stem cell factor (SCF) at a concentration of 50 ng/mL resulted in the most significant increase in cell proliferation and HbF content. The average HbF/hemoglobin A (HbA) ratio was 30.9% ؎ 18.7% in cultures containing SCF compared with 4.1% ؎ 2.2% in those grown with erythropoietin (EPO) alone (P ‫؍‬ 8.5E-8). To further investigate the hemoglobin-modulating effects of SCF, we examined the surface expression pattern of the SCF receptor, CD117, among maturing erythroblasts. CD117 expression increased during the first week of culture and peaked on culture days 7 to 9. After culture day 9, the level of CD117 declined to lower levels. The rise in CD117 expression to high levels mirrored that of the transferrin receptor (CD71), and the subsequent reduction in CD117 was inversely related to increases in expression of glycophorin A. SCF-related increases in the HbF/HbA ratio correlated with the expression pattern of CD117. SCF added during days 7 to 14 resulted in a more pancellular distribution of HbF on day 14 compared with the heterocellular distribution present in cultures supplemented with SCF on days 0 to 7. A significant SCF-mediated increase in HbF was also measured using progenitors derived from cord blood. These results suggest that the HbF response to SCF is greatest at the late progenitor stage as a function of surface CD117 expression. (Blood. 2003;

Exposure to nanosilver found in food-and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food-and cosmetics-related chemicals including nanosilver. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.