Demet Nalbant

The Glc7 Phosphatase Subunit of the Cleavage and Polyadenylation Factor Is Essential for Transcription Termination on snoRNA Genes

Glc7, the yeast protein phosphatase 1, is a component of the cleavage and polyadenylation factor (CPF). Here we show that downregulation of Glc7, or its dissociation from CPF in the absence of CPF subunits Ref2 or Swd2, results in similar snoRNA termination defects. Overexpressing a C-terminal fragment of Sen1, a superfamily I helicase required for snoRNA termination, suppresses the growth and termination defects associated with loss of Swd2 or Ref2, but not Glc7. Suppression by Sen1 requires nuclear localization and direct interaction with Glc7, which can dephosphorylate Sen1 in vitro. The suppressing fragment, and in a similar manner full-length Sen1, copurifies with the snoRNA termination factors Nrd1 and Nab3, suggesting loss of Glc7 from CPF can be compensated by recruiting Glc7 to Nrd1-Nab3 through Sen1. Swd2 is also a subunit of the Set1c histone H3K4 methyltransferase complex and is required for its stability and optimal methyltransferase activity.

Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin densities

BACKGROUND: Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 (51Cr)‐labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs).

A Comparative Study of Drosophila and Human A-Type Lamins

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm0, which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction.

Evidence of receptor-mediated elimination of erythropoietin by analysis of erythropoietin receptor mRNA expression in bone marrow and erythropoietin clearance during anemia.

Erythropoietin (Epo) is the primary hormone that stimulates erythroid proliferation and differentiation through its cell surface receptor (EpoR) on erythroid progenitor cells. Previous studies have suggested that the bone marrow plays an important role in Epos elimination. The changes in the EpoR mRNA levels and Epos clearance in the bone marrow of 11 newborn lambs were studied to elucidate the role of EpoR in Epos clearance under anemic conditions. Epo mRNA levels were measured by real-time polymerase chain reaction, and relative expression of EpoR was calculated by using the comparative CT method. The glyceraldehyde-3-phosphate dehydrogenase housekeeping gene was chosen as a control gene for the calculations. All lambs showed significant increase in bone marrow EpoR mRNA levels after phlebotomy-induced anemia. Epos clearance determined from simultaneous pharmacokinetic studies with 125I-recombinant human Epo showed a significant increase after phlebotomy-induced anemia that was similar to the increase in EpoR. By day 28 after phlebotomy, EpoR mRNA levels and Epo clearance had returned toward baseline. These results indicate that the changes in Epos clearance are not caused by body growth but result from significant changes in the pool of EpoR. A linear mixed-effect model was used to evaluate the quantitative relationship between EpoR and Epos clearance. This analysis demonstrated a highly significant positive linear correlation between EpoR and Epo clearance. Together, these findings provide strong evidence that receptor-mediated Epo clearance is an important route for Epos elimination.

Red blood cell (RBC) volume can be independently determined in vivo in humans using RBCs labeled at different densities of biotin

BACKGROUND: Anemia is a serious problem in critically ill neonates. To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of red blood cell volume (RCV) and blood volume is useful. To extend our previous sheep study in which RBCs were labeled at four different biotin densities, we assessed the validity of this multidensity method for in vivo measurement of circulating RCV in humans.

A mathematical modeling approach to quantify the role of phlebotomy losses and need for transfusions in neonatal anemia

BACKGROUND: Very preterm infants commonly develop anemia requiring multiple red blood cell transfusions (RBCTx). This is in part attributable to heavy laboratory phlebotomy loss. Quantification of the extent to which laboratory blood loss contributes to anemia sufficient to prompt RBCTx has not been examined.

Comparison of multiple red cell volume methods performed concurrently in premature infants following allogeneic transfusion

Background:Study of the pathophysiology and treatment of anemia of prematurity is facilitated by direct measurement of red cell volume (RCV) utilizing microliter quantities of blood samples. Our objective was to compare concurrent measurements of multiple direct RCV methods in infants.Methods:Eighteen preterm infants receiving clinically indicated transfusions had concurrent flow cytometric determinations of RCV and 24-h red blood cell (RBC) recovery based on donor–recipient differences of biotin-labeled RBCs (BioRBCs), Kidd antigen mismatched RBCs, and fetal hemoglobin–positive (HbF+) RBCs. High-performance liquid chromatography (HPLC) was also used for measuring HbF and adult hemoglobin protein concentrations for the determination of RCV.Results:Concurrent RCV measurements using BioRBCs (18 and 54 µg/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared with RCVs measured using the reference BioRBC density (6 µg/ml). By contrast, the HbF-HPLC method overestimated RCV by 45% compared with the reference method. All the methods demonstrated 100% 24-h posttransfusion RBC recovery (PTR24).Conclusion:Because BioRBC, Kidd antigen, and fetal hemoglobin (HbF) flow cytometry are safe and accurate methods requiring <10 µl of patient blood for determining RCV and PTR24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.

Red blood cell (RBC) volume can be independently determined in vivo in the sheep using ovine RBCs labeled at different densities of biotin.

BACKGROUND: To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin, repeated measurement of the circulating red blood cell volume (RCV) would be useful. Ovine erythropoiesis is similar to human erythropoiesis. Accordingly, a method for measuring RCV using either human or sheep RBCs labeled at different biotin densities has been previously validated in vitro. Here preclinical studies validating this method for in vivo measurement of circulating RCV in sheep are reported.

Population pharmacodynamic analysis of erythropoiesis in preterm infants for determining the anemia treatment potential of erythropoietin.

A population pharmacokinetics/pharmacodynamic (PK/PD) model was developed to describe changes in erythropoiesis as a function of plasma erythropoietin (EPO) concentration over the first 30 days of life in preterm infants who developed severe anemia requiring red blood cell (RBC) transfusion. Several covariates were tested as possible factors influencing the responsiveness to EPO. Discarded blood samples in 27 ventilated preterm infants born at 24-29 wk of gestation were used to construct plasma EPO, hemoglobin (Hb), and RBC concentration-time profiles. The amount of Hb removed for laboratory testing and that transfused throughout the study period were recorded. A population PK/PD model accounting for the dynamic Hb changes experienced by these infants was simultaneously fitted to plasma EPO, Hb, and RBC concentrations. A covariate analysis suggested that the erythropoietic efficacy of EPO is increased for preterm infants at later gestational ages. The PD analysis showed a sevenfold difference in maximum Hb production rate dependent on gestational age and indicated that preterm infants, when stimulated by EPO, have the capacity to produce additional Hb that may result in a decrease in RBC transfusions. The present model has utility in clinical trial simulations investigating the treatment potential of erythropoietic stimulating agents in the treatment of anemia of prematurity.

Mean remaining life span: a new clinically relevant parameter to assess the quality of transfused red blood cells.

The quality of transfused red blood cells (RBCs) to treat anemia depends on its potential for oxygen delivery, governed by two properties: 1) initial posttransfusion recovery and 2) life span of initially surviving RBCs. The latter property is poorly evaluated by the traditional mean potential life span (MPL) or mean cell age (MA), because these parameters do not evaluate how long transfused RBCs remain in circulation. Furthermore, evaluation of MPL is based on two problematic assumptions regarding transfused RBCs: 1) they were produced at a constant steady‐state rate and 2) they have similar storage life spans.