[3] Detection of globin chains by reversed-phase high-performance liquid chromatography

Masala, Bruno Lucio; Manca, Laura

doi:10.1016/0076-6879(94)31005-x

Cited by 50 publications

(25 citation statements)

References 60 publications

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“…For all samples, cell debris was removed via centrifugation at 10,000g for 30 min at 4°C and dialyzed in 10 mM sodium/potassium phosphate, pH 7.0, overnight. The final hemoglobin concentration was determined based on the absorbance of the ␣-band such that absorbance ϫ dilution ϫ 1.14 ϭ mg/mL (16). Oxidation to ferric hemoglobin was monitored based on the ratio of absorbance of the ␣ and ␤ bands.…”

Section: Methodsmentioning

confidence: 99%

Highly Reactive Cysteine Residues in Rodent Hemoglobins

Miranda

2000

Biochemical and Biophysical Research Communications

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Section: Methodsmentioning

confidence: 99%

Highly Reactive Cysteine Residues in Rodent Hemoglobins

Miranda

2000

Biochemical and Biophysical Research Communications

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“…Packed erythroid cells composed of reticulocytes and RBC 1 (Ͼ99.4%) were washed three times in 1ϫ Dulbecco's PBS and incubated in 1 ml of [ 3 H]leucine labeling mix (18) supplemented with 50 g of transferrin for 1, 2, and 2.5 h with periodic shaking under 5% CO 2 in air (15). The RBC were then lysed, and the hemoglobin solution was quantified as previously described (19). The hemoglobin solution (100 g) was used to determine the globin chain content by high pressure liquid chromatography on a Vydac large pore C 4 column (4.6 ϫ 250 mm; Grace Vydac, Hesperia, CA).…”

Section: Globin Chain Analysismentioning

confidence: 99%

Differential Regulatory and Compensatory Responses in Hematopoiesis/Erythropoiesis in α- and β-Globin Hemizygous Mice

Beauchemin¹,

Blouin

Trudel

2004

Journal of Biological Chemistry

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Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in ␣-and ␤-globin hemizygous mice, lacking the two adult tandemly organized genes from either the ␣-or ␤-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, ␤-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for ␣-globin hemizygous mice. In contrast, however, ␤-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of ␤-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free ␣-chain than ␤-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the ␤-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in ␤-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units ϳ100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units ϳ7-fold). This early compensatory mechanism was less pronounced in ␣-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for ␣-and ␤-thalassemia.

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“…were determined by the reversed-phase HPLC method, as previously described [22]. DNA extraction and Sutton's haplotype of the ␤-globin gene cluster were performed as reported for nine restriction fragment length polymorphisms (RFLPs) in the ␤-gene complex: [23,24].…”

Section: Materials and Methods Subjectsmentioning

confidence: 99%

HbF production in β thalassaemia heterozygotes for the IVS-II-1 G→A β0-globin mutation. Implication of the haplotype and theGγ-158 C→T mutation on the HbF level

et al. 2000

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We studied the presence of the XmnI site and the ␤-globin haplotype in 24 individuals, carriers of the IVSII-1 G→A ␤ 0 -globin mutation, of whom fourteen had no detectable levels of HbF, while ten coming from 5 families, presented HbF levels ranging from 1.7 to 9% of the total Hb. Of these ␤-thalassaemia heterozygotes with fetal hemoglobin, 6 were females and 4 were males with median HbF levels of 4.85% and 4% respectively, and an excess of G

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[3] Detection of globin chains by reversed-phase high-performance liquid chromatography

Cited by 50 publications

References 60 publications

Highly Reactive Cysteine Residues in Rodent Hemoglobins

Highly Reactive Cysteine Residues in Rodent Hemoglobins

Differential Regulatory and Compensatory Responses in Hematopoiesis/Erythropoiesis in α- and β-Globin Hemizygous Mice

HbF production in β thalassaemia heterozygotes for the IVS-II-1 G→A β0-globin mutation. Implication of the haplotype and theGγ-158 C→T mutation on the HbF level

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