Expression of alpha-hemoglobin stabilizing protein gene during human erythropoiesis

alpha-Hemoglobin stabilizing protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free alpha-hemoglobin but not with beta-hemoglobin or hemoglobin A. As such, AHSP is required for normal erythropoiesis, probably acting by blocking the deleterious effects of free alpha-hemoglobin precipitation. In order to study the levels of expression of the AHSP gene during the different phases of erythropoiesis, we carried out a two-phase liquid culture of erythroid cells and real-time quantitative polymerase chain reaction. Blood from control volunteers was cultured with erythropoietin to stimulate differentiation. The different stages of erythropoiesis were confirmed by morphologic and flow cytometric analysis. The results showed a progressive increase in AHSP gene expression following the expression of alpha-globin gene, during maturation of the red blood cell precursors, confirming the probable important function of this protein during normal erythropoiesis. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.32215716

beta-Spectrin S-ta Barbara: a novel frameshift mutation in hereditary spherocytosis associated with detectable levels of mRNA and a germ cell line mosaicism

Hereditary spherocytosis (HS) is a common inherited anaemia characterized by the presence of spherocytic red cells and by a heterogeneous nature in terms of its clinical presentation, molecular basis and inheritance. Defects in several membrane protein genes have been involved in the pathogenesis of HS, including defects in the beta -spectrin gene. We detected a novel frameshift mutation in the beta -spectrin gene, a C deletion at codon 638, in a patient presenting with HS and spectrin deficiency. The mutant protein was not detected in the membrane or in other cellular compartments, but detectable levels of mutant mRNA were found in the patient. Interestingly, this mutation was not present in the patient's parents, suggesting a genetic mosaicism, especially as the patient has an affected brother with the same molecular defect. We analysed DNA from different tissues of the parents and the mutation was absent from all tissues analysed. This mutation seems to be confined to the germ cell lineage of the patient's mother and must present a mosaic pattern in these cells as the patient also has unaffected siblings.115234735

Regulation of Chondrogenesis by Transforming Growth Factor-beta 3 and Insulin-like Growth Factor-1 from Human Mesenchymal Umbilical Cord Blood Cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objective. Mature articular cartilage is vulnerable to injuries and disease processes that cause irreversible tissue damage because of its limited capacity for self-repair. Umbilical cord blood is a source of mesenchymal stem cells, which can give rise to cells of different lineages, including cartilage, bone, and fat. Cellular condensation is a required step in the initiation of mesenchymal chondrogenesis. We attempted to differentiate cells from umbilical cord blood into chondrocytes with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta 3 (TGF-beta 3). Methods. Cells were grown in high density micromass and monolayer culture systems and then evaluated for expression of type 11 collagen, aggrecan, and Sox9. Umbilical cord blood from 130 patients was harvested. Results. Expression of type II collagen, aggrecan, and Sox9 was detected after 14 days in TGF-beta 3- and IGF-1-stimulated cells in both types of culture (monolayer and micromass). On Day 21 in the micromass culture, expression levels were greater than they were at 14 days for all genes. TGF-beta 3 was found to be more efficient at promoting chondrogenesis than IGF-1. By western blot, we also found that after 3 weeks, the expression of type II collagen was greater in micromass culture with TGF-beta 3. Conclusion. TGF-beta 3 used in micromass culture is the best growth factor for promoting the proliferation and differentiation of mesenchymal cells from umbilical cord blood during chondrogenesis. This approach may provide an alternative to autologous grafting. (First Release May 15 2010; J Rheumatol 2010;37:1519-26; doi: 10.3899/jrheum.091169)37715191526Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Chondrogenesis from umbilical cord blood cells stimulated with BMP-2 and BMP-6

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Umbilical cord blood contains undifferentiated mesenchymal stem cells (MSCs) with chondrogenic potential that may be used for the repair of joint damage. The role of growth factors during the process of chondrogenesis is still not entirely understood. The objective of this study was to evaluate the formation of chondrocytes, cartilaginous matrix and type II collagen from human umbilical cord blood stem cells exposed to two different growth factors, BMP-6 and BMP-2, while being cultured as a micromass or a monolayer. Umbilical cord blood was obtained from full-term deliveries, and then, mononuclear cells were separated and cultured for expansion. Afterward, these cells were evaluated by flow cytometry using antibodies specific for MSCs and induced to chondrogenic differentiation in micromass and monolayer cultures supplemented with BMP-2 and BMP-6. Cellular phenotype was evaluated after 7, 14 and 21 days by RT-PCR and Western blot analysis to identify the type II collagen and aggrecan. The expanded cells displayed surface antigens characteristic of mesenchymal progenitor cells and were negative for hematopoietic differentiation antigens. Type II collagen and aggrecan mRNAs were expressed from day 14 in cells stimulated with BMP-2 or BMP-6. Type II collagen was demonstrated by Western blotting in both groups, and the greatest expression was observed 21 days after the cells were stimulated with BMP-2 cultured in micromass. BMP-2 in micromass culture was more efficient to induce the chondrogenesis.331121128Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ANKHD1 is a multiple ankyrin repeat containing protein, highly expressed in cancers, such as acute leukemia. The present study was undertaken to determine the expression and functional significance of ANKHD1 in human Multiple Myeloma (MM). We found that ANKHD1 is highly expressed in MM patient cells and cell lines. In vitro, lentiviral mediated ANKHD1-shRNA inhibited proliferation and delayed S to G2M cell cycle progression in glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further ANKHD1 silencing resulted in upregulation of cyclin dependent kinase inhibitor p21 irrespective of the p53 status of the MM cell lines. These data suggest that ANKHD1 might have a role in MM cell proliferation and cell cycle progression by regulating expression of p21. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.5862443114318Instituto Nacional de Ciencia e Tecnologia do Sangue(INCTS)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Molecular characterization of hemoglobin alpha-D chains from Geochelone carbonaria and Geochelone denticulata land turtles

In order to help elucidate the evolution of alpha-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles alpha-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb alpha-chain, alterations in important regions can be noted: alpha110 Ala-Gly, alpha114 Pro-Gly, alpha117 Phe-Tyr and alpha122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken alpha-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish alpha-chains. Phylogenetic analysis of alpha-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. (C) 2002 Elsevier Science Inc. All rights reserved.134238939

DNAase I hypersensitive site 3 ' to the beta-globin gene cluster contains a TAA Insertion specific for beta(s)-Benin haplotype

Background and Objectives. Analysis of DNA polymorphic sites is a powerful tool for detection of gene flow in human evolutionary studies and to trace genetic background associated with abnormal genes. The beta-globin focus contains more than 20 single-base restriction fragment length polymorphism (RFLP) sites spanning over 80 kb on chromosome 11. Far downstream of the expressed genes, there is a hypersensitive site (HS). The function of the 3'-HS remains unknown. As an approach to the understanding of the 3'-HS region in sickle cell anemia we searched for sequence polymorphism in the AT-rich region, using a non-radioactive polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) technique. Design and Methods. A 460 bp fragment located at the 3' of the beta globin gene was amplified from patients (with sickle cell anemia and HbSC disease), and from AS individuals. Standard RFLP-haplotyping was performed and compared with the PCR-SSCP screening strategy. Results. Two distinct band patterns were revealed by SSCP testing, each one in strict linkage disequilibrium with either Benin or Bantu haplotypes. Direct sequencing of the amplified segment revealed a TAA insertion in the AT-rich region, in all 121 beta(s) Benin chromosomes tested, but not in other beta(s) haplotypes from the total of 380 beta(s) chromosomes typed. Interpretation and Conclusions. SSCP analysis could easily distinguish sequence variations in the XAT-rich region of the beta-globin cluster, and a TAA insertion in this region seems to be specific for the Benin-beta(s) chromosome. (C) 2002, Ferrata Storti Foundation.87324624

Viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection

Background: Umbilical cord blood (UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples. Study Design and Methods: Two experiments were performed: in the first one, volume reduction of the UCB units was carried out before analysis. In the second one, analysis was carried out with no previous manipulation. Samples were stored at room temperature and one aliquot was taken daily for analysis. We examined CD34+ cell, B-cell precursor, mature B and T lymphocyte, monocyte, granulocyte, and mesenchymal stem cell (MSCs) concentrations. Results: Thirty-six UCB units were analyzed. CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit (CFU) number in UCB units stored for 96 hours. Conclusion: UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection. CD34+ cells and T lymphocytes increased, probably due to the loss of other subsets. CFU growth during the period analyzed and confirmed stem cell functionality, despite the decrease at 96 hours. Results demonstrated that UCB units could probably be processed up to 96 hours after collection.5392034204

Molecular characterization of hemoglobin alpha-D chains from Geochelone carbonaria and Geochelone denticulata land turtles

In order to help elucidate the evolution of alpha-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles alpha-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb alpha-chain, alterations in important regions can be noted: alpha110 Ala-Gly, alpha114 Pro-Gly, alpha117 Phe-Tyr and alpha122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken alpha-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish alpha-chains. Phylogenetic analysis of alpha-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. (C) 2002 Elsevier B.V. All rights reserved

Expression of peroxiredoxins I and IV in multiple myeloma: association with immunoglobulin accumulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)B cell malignancies are classified according to the postulated differentiation stage of the originating cell. During differentiation, structural and molecular changes occur to support massive processing of immunoglobulin in the endoplasmic reticulum (ER) of plasma cells at the final stage. When overloaded, the ER generates unfolded proteins and hydrogen peroxide (H2O2), which may cause cell death. Peroxiredoxins (Prxs) I and IV belong to a family of proteins able to catalyze peroxide detoxification. Here, we investigated a potential association of these enzymes with immunoglobulin production in B cell neoplasms. Our results demonstrated that the expression of Prx IV was induced as cells became competent to synthesize immunoglobulin light chains, as observed by immunohistochemistry in tissue sections of B cell neoplasms and also by qPCR and Western blotting analyses in malignant B cell lines. Prx I was frequently highly expressed, indicating additional regulatory processes besides ER activity. Results obtained exclusively with myeloma cells have shown that expression of Prxs I and IV, both at mRNA and protein levels, was associated with light chain secretion quantified by ELISA. We suggest that Prxs I and IV may provide survival advantages for terminally differentiated neoplastic B cells by the elimination of H2O2 and, in the case of Prx IV, by the conversion of this toxic in a functional agent driving oxidative protein folding in the ER. In this sense, multiple myeloma and lymphomas demonstrated to synthesize immunoglobulin chains may benefit from strategic therapies targeting the adaptive pathway to ER stress, including inhibition of Prxs I and IV activity.46314755Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP