ABSTRACT
Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.
Subject(s)
Anemia, Sickle Cell , Disease Models, Animal , Mice, Transgenic , Animals , Hemoglobin, Sickle/genetics , Humans , MiceABSTRACT
Use of hydroxyurea (HU) to treat sickle cell disease is usually associated with increments in fetal hemoglobin (Hb F) production; however, in vitro studies show that HU may also induce hemoglobin denaturation. Whole blood samples from Hb AA, Hb AS, and Hb SS patients were treated in vitro with 100, 150, 200, 250, and 300 micrograms/mL HU, incubated at 30 degrees C for up to 12 days, and analyzed by high-performance liquid chromatography (HPLC). Hb AA levels show decrements of 91 to 14% with 100 micrograms/mL and 89 to 4% with 150 micrograms/mL after 12 days; 86 to 2% with 200 micrograms/mL after 10 days; 86 to 8% with 250 and 300 micrograms/mL after 8 days. Similar treatment and incubation times for Hb AS whole blood demonstrate that HU equally degrades the A and S components of Hb AS. A comparable approach for Hb SS whole blood samples, using a 300 micrograms/mL HU treatment, showed a hemoglobin denaturing pattern that went from 93% to 1% after 12 days. Globin chain analysis of these samples by reverse-phase HPLC showed that the denaturing effects occur mostly on the beta-globin chain.
Subject(s)
Antisickling Agents/adverse effects , Hemoglobin, Sickle/drug effects , Hemoglobins/drug effects , Hydroxyurea/adverse effects , Adolescent , Adult , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/drug therapy , Hemoglobin A/chemistry , Hemoglobin A/drug effects , Hemoglobin, Sickle/chemistry , Hemoglobins/chemistry , Humans , In Vitro Techniques , Male , Protein Denaturation/drug effectsABSTRACT
Nucleoids isolated from either synchronized or exponentially growing HeLa cells were digested with restriction enzymes to separate a nuclear matrix-bound DNA component from the rest. Partial libraries were constructed by inserting DNA fragments from both components into a yeast-bacteria plasmid vector. A random sample from these libraries was tested for ARS activity by a standard yeast transformation assay. We found that synchronization for DNA replication results in an enrichment for autonomously replicating sequences in the library constructed with the DNA component bound to the nuclear matrix.
