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The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene. We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies. This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.

Abstract 4243 Feline Leukemia Virus subgroup C Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukemia virus subgroup C, causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter which is essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells1. Moreover, Keel and co-authors have reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis; also post-natal mice lacking FLVCR showed severe anemia. In addition to the erythroid defect, Flvcr-null embryos display defective growth and developmental anomalies2. We have identified an alternative transcription start site giving rise to a novel FLVCR isoform (FLVCRb). Flvcr-b transcript completely lacks the first exon of the canonical isoform (FLVCRa) and code for a putative 6 transmembrane domain containing protein ubiquitously expressed. In vitro over-expression of FLVCRa and FLVCRb showed that the two proteins display different subcellular localization. As expected, FLVCRa is localized at the cell membrane while FLVCRb is in the mitochondrial compartment. The mitochondrial localization of this novel isoform is further confirmed by the identification of a N-terminal mitochondrial sorting presequence. The mitochondrion is the site in which heme biosynthesis occurs. Although all the enzymatic reactions involved in heme synthesis are well characterized, how heme is exported to the cytosol is largely unknown. Because of FLVCRa is a heme exporter at the cell membrane, we hypothesized that FLVCRb could be the mitochondrial heme exporter. According to this hypothesis, FLVCRb expression increased following the stimulation of heme biosynthesis in vitro, in correlation with the increase in hemoglobin production. The ability of FLVCRb to bind and export heme out of the mitochondria is still under investigation. To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported2. Keel and co-author generated a mouse model in which both FLVCRa and FLVCRb have been deleted. In our mouse model, FLVCRa has been specifically deleted while FLVCRb is still expressed (FLVCRa-null mice). Flvcr-a +/− mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a +/− mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained. The analysis of the embryos from timed Flvcr-a +/− intercrosses showed that the Flvcr-a homozygous knock-out genotype was lethal between E14.5 and the birth. E13.5 Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Imcomplete vasculogenesis in the Flvcr-a-null embryos was observed at E11.5, a developmental stage in which hemorrhages were not still evident. This suggests that hemorrhages arise from a defect in the development of embryonic vasculature. Moreover, FLVCRa-null embryos showed skeletal abnormalities as demonstrated by Alcian blue-alizarin red staining. Skeletal malformations were evident in the limb where digits did not form properly and in the head where Meckel's cartilage was incomplete. It is interesting to note that this kind of malformations also occurs in Diamond Blackfan Anemia (DBA) patients. Surprisingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) showed normal erythropoiesis in Flvcr-a-null embryos, in opposition to what occurs in the previously reported Flvcr-null mice2. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis when the expression of FLVCRa is loss, likely exporting heme out of the mithocondrion for hemoglobin synthesis. Moreover, the loss of FLVCRa leads to incomplete vasculogenesis, hemorrhages and skeletal malformations highlighting new roles of FLVCRa in these processes. 1. Quigley JG et al. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004 2. Keel SB et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008. Disclosures: No relevant conflicts of interest to declare.

Abstract 1080 Poster Board I-102 Feline Leukemia Virus, subgroup C, Receptor (FLVCR) was originally identified and cloned as a cell-surface protein receptor for feline leukaemic virus, subgroup C causing pure red blood cell aplasia in cats. Recent studies have demonstrated that FLVCR is a heme exporter essential for erythropoiesis. The heme efflux via FLVCR was shown to be essential for erythroid differentiation in K562 cells as well as in CD34+ precursors cells1. Moreover, Keel and co-authors have recently reported that Flvcr-null mice die in utero due to the failure of fetal erythropoiesis2. We have recently identified a novel FLVCR isoform, called FLVCRb, coding for a putative 7 transmembrane domain-containing protein. Both FLVCRb and the canonical FLVCR (described herein as FLVCRa) were expressed in all tissues: FLVCRa mRNA level was significantly higher than FLVCRb in liver, kidney, colon and duodenum, whereas the opposite was observed in heart and skeletal muscle. In bone marrow, spleen and brain, FLVCRa and FLVCRb mRNA levels were comparable. FLVCRa and FLVCRb display different sub-cellular distribution when over-expressed in vitro. As expected, FLVCRa is localized at the cell membrane while FLVCRb is expressed in intracellular compartment suggesting different functions of the two proteins in heme metabolism. To gain insights into the specific roles of the two isoforms, we have generated Flvcr mutant mice different from those previously reported2. Keel and co-author generated a mouse model in which both FLVCRa and FLVCRb were deleted. In our mouse model, FLVCRa has been specifically deleted and FLVCRb is still expressed (Flvcr-a-null mice). Flvcr-a heterozygous mice were grossly normal, fertile and indistinguishable from their wild-type littermates. When Flvcr-a +/- mice were intercrossed, no Flvcr-a homozygous knock-out newborns were obtained, and the analysis of the embryos from timed Flvcr-a +/- intercrosses showed that the Flvcr-a-/- genotype was lethal between E14.5-E16.5. Flvcr-a-null embryos showed multifocal and extended hemorrhages, visible in the limbs, head and throughout the body wall, as well as subcutaneous edema. Alcian blue-alizarin red staining demonstrated skeletal abnormalities in limbs and head similar to that observed in Diamond-Blakfan anemia (DBA) patients. Interestingly, flow cytometric analyses of E14.5 fetal liver cells double-stained for Ter119 (erythroid-specific antigen) and CD71 (transferrin receptor) show normal erythropoiesis in Flvcr-a-null embryos, opposite to the previously reported Flvcr-null mice2. Taken together, these data demonstrated that FLVCRb is sufficient to support fetal erythropoiesis, but not to prevent endothelial ruptures responsible for hemorrhages, thus suggesting that FLVCRa is needed for detoxifying heme excess at these sites. The involvement of the two isoforms in the pathogenesis of DBA is currently under investigation. Quigley JG, Yang Z, Worthington MT, Phillips JD, Sabo KM, Sabath DE, Berg CL, Sassa S, Wood BL, Abkowitz JL. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004;118:757-66. Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, Kingsley PD, De Domenico I, Vaughn MB, Kaplan J, Palis J, Abkowitz JL. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825-8. Disclosures No relevant conflicts of interest to declare.