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A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.

ABSTRACT: Hemoglobin (Hb) E (?26 Glu?Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess ?-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid ?(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of ?(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC ?/? (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess ?-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

SUBMITTER: Chen Q

PROVIDER: S-EPMC3310900 | biostudies-other | 2012 Feb

REPOSITORIES: biostudies-other

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A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.

Chen Qiuying Q Fabry Mary E ME Rybicki Anne C AC Suzuka Sandra M SM Balazs Tatiana C TC Etzion Zipora Z de Jong Kitty K Akoto Edna K EK Canterino Joseph E JE Kaul Dhananjay K DK Kuypers Frans A FA Lefer David D Bouhassira Eric E EE Hirsch Rhoda Elison RE

Blood cells, molecules & diseases 20120118 2

Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical fo ...[more]

PMID: 22260787

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Project description:BackgroundCancer is associated with muscle atrophy (cancer cachexia) that is linked to up to 40% of cancer-related deaths. Oxidative stress is a critical player in the induction and progression of age-related loss of muscle mass and weakness (sarcopenia); however, the role of oxidative stress in cancer cachexia has not been defined. The purpose of this study was to examine if elevated oxidative stress exacerbates cancer cachexia.MethodsCu/Zn superoxide dismutase knockout (Sod1KO) mice were used as an established mouse model of elevated oxidative stress. Cancer cachexia was induced by injection of one million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (saline) into the hind flank of female wild-type mice or Sod1KO mice at approximately 4 months of age. The tumour developed for 3 weeks. Muscle mass, contractile function, neuromuscular junction (NMJ) fragmentation, metabolic proteins, mitochondrial function, and motor neuron function were measured in wild-type and Sod1KO saline and tumour-bearing mice. Data were analysed by two-way ANOVA with Tukey-Kramer post hoc test when significant F ratios were determined and α was set at 0.05. Unless otherwise noted, results in abstract are mean ±SEM.ResultsMuscle mass and cross-sectional area were significantly reduced, in tumour-bearing mice. Metabolic enzymes were dysregulated in Sod1KO mice and cancer exacerbated this phenotype. NMJ fragmentation was exacerbated in tumour-bearing Sod1KO mice. Myofibrillar protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.00847 ± 0.00205; wildtype LLC, 0.0211 ± 0.00184) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0180 ± 0.00118; Sod1KO LLC, 0.0490 ± 0.00132). Muscle mitochondrial oxygen consumption was reduced in tumour-bearing mice compared with saline-injected wild-type mice. Mitochondrial protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.0204 ± 0.00159; wild-type LLC, 0.167 ± 0.00157) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0231 ± 0.00108; Sod1 KO LLC, 0.0645 ± 0.000631). Sciatic nerve conduction velocity was decreased in tumour-bearing wild-type mice (wild-type saline, 38.2 ± 0.861; wild-type LLC, 28.8 ± 0.772). Three out of eleven of the tumour-bearing Sod1KO mice did not survive the 3-week period following tumour implantation.ConclusionsOxidative stress does not exacerbate cancer-induced muscle loss; however, cancer cachexia may accelerate NMJ disruption.

Project description:In this work, we discuss the development of H.O.S.T., a novel hemoglobin microbubble-based electrochemical biosensor for label-free detection of Hydrogen peroxide (H2O2) towards oxidative stress and cancer diagnostic applications. The novelty of the constructed sensor lies in the use of a sonochemically prepared hemoglobin microbubble capture probe, which allowed for an extended dynamic range, lower detection limit, and enhanced resolution compared to the native hemoglobin based H2O2 biosensors. The size of the prepared particles Hemoglobin microbubbles was characterized using Coulter Counter analysis and was found to be 4.4 microns, and the morphology of these spherical microbubbles was shown using Brightfield microscopy. The binding chemistry of the sensor stack elements of HbMbs' and P.A.N.H.S. crosslinker was characterized using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy and UV-Vis Spectroscopy. The electrochemical biosensor calibration (R2 > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The electrochemical biosensor calibration (R2 > 0.95) was done using Electrochemical Impedance Spectroscopy, Cyclic Voltammetry, and Square Wave Voltammetry. The specificity of the sensor for H2O2 was analyzed using cross-reactivity studies using ascorbic acid and glucose as interferents (p < 0.0001 for the highest non-specific dose versus the lowest specific dose). The developed sensor showed good agreement in performance with a commercially available kit for H2O2 detection using Bland Altman Analysis (mean bias = 0.37 for E.I.S. and - 24.26 for CV). The diagnostic potential of the biosensor was further tested in cancerous (N.G.P.) and non-cancerous (H.E.K.) cell lysate for H2O2 detection (p = 0.0064 for E.I.S. and p = 0.0062 for CV). The Michaelis Menten constant calculated from the linear portion of the sensor was found to be [Formula: see text] of 19.44 µM indicating that our biosensor has a higher affinity to Hydrogen peroxide than other available enzymatic sensors, it is attributed to the unique design of the hemoglobin polymers in microbubble.

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A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.

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A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.

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