Biomedical subjects

Luanne L Peters

Publications and source records attributed to Luanne L Peters.

At least 19 recordsLinked to original sources

PubMed2007

The mouse as a model for human biology: a resource guide for complex trait analysis.

The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits.

Animals↗

PubMed2006

Combined deletion of mouse dematin-headpiece and beta-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia.

Dematin and adducin are actin-binding proteins of the erythrocyte "junctional complex." Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking beta-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an approximately 12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.

Actins↗

PubMed2006

Novel method for high-throughput phenotyping of sleep in mice.

Assessment of sleep in mice currently requires initial implantation of chronic electrodes for assessment of electroencephalogram (EEG) and electromyogram (EMG) followed by time to recover from surgery. Hence, it is not ideal for high-throughput screening. To address this deficiency, a method of assessment of sleep and wakefulness in mice has been developed based on assessment of activity/inactivity either by digital video analysis or by breaking infrared beams in the mouse cage. It is based on the algorithm that any episode of continuous inactivity of > or =40 s is predicted to be sleep. The method gives excellent agreement in C57BL/6J male mice with simultaneous assessment of sleep by EEG/EMG recording. The average agreement over 8,640 10-s epochs in 24 h is 92% (n = 7 mice) with agreement in individual mice being 88-94%. Average EEG/EMG determined sleep per 2-h interval across the day was 59.4 min. The estimated mean difference (bias) per 2-h interval between inactivity-defined sleep and EEG/EMG-defined sleep was only 1.0 min (95% confidence interval for mean bias -0.06 to +2.6 min). The standard deviation of differences (precision) was 7.5 min per 2-h interval with 95% limits of agreement ranging from -13.7 to +15.7 min. Although bias significantly varied by time of day (P = 0.0007), the magnitude of time-of-day differences was not large (average bias during lights on and lights off was +5.0 and -3.0 min per 2-h interval, respectively). This method has applications in chemical mutagenesis and for studies of molecular changes in brain with sleep/wakefulness.

Algorithms↗

PubMed2006

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes.

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1(-/-); Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1(-/-) mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1(-/-) erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 +/- 3.2 in 4.1(-/-) vs. 9.8 +/- 1.3 mmol/10(13) cell x h in control mice), with an abnormal dependence on osmolality (EC(50) = 417 +/- 42 in 4.1(-/-) vs. 460 +/- 35 mosmol/kgH(2)O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K(0.5) = 489.7 +/- 0.7 vs. 537.0 +/- 0.56 nM in control mice), the V(max) of the H-induced Na/H exchange activity was markedly elevated in 4.1(-/-) erythrocytes (V(max) 91.47 +/- 7.2 compared with 46.52 +/- 5.4 mmol/10(13) cell x h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1(-/-) erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1(-/-) erythrocytes.

Animals↗

PubMed2006

Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion.

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis.

Actins↗

PubMed2006

A mouse TRAPP-related protein is involved in pigmentation.

We identified a new spontaneous recessive mutation in the mouse, mhyp (mosaic hypopigmentation), in a screen for novel proviral integration sites in a multiple ecotropic provirus mapping stock. Integration of an 8.4-kb retrovirus results in mosaic loss of coat pigment in mhyp homozygotes. Patchy loss of pigmentation in the retinal pigmented epithelial layer of the eye with abnormal melanosomes is also evident. We mapped mhyp to mouse chromosome 7 and cloned the underlying gene. mhyp is a defect in the Trappc6a gene. Expression of Trappc6a is markedly diminished in mhyp homozygotes. The normal protein, TRAPPC6A, is a subunit of the TRAPP (transport protein particle) I and II complexes. While TRAPP complexes are essential for ER-to-Golgi and intra-Golgi vesicle trafficking in yeast, TRAPP subunits participate in additional, including post-Golgi, transport events in mammals. The data implicate mammalian TRAPPC6A in vesicle trafficking during melanosome biogenesis.

Amino Acid Sequence↗

PubMed2006

Quantitative trait loci for baseline erythroid traits.

A substantial genetic contribution underlies variation in baseline peripheral blood counts. We performed quantitative trait locus/loci (QTL) analyses to identify chromosome (Chr) regions harboring genes influencing the baseline erythroid parameters in F2 intercrosses between NZW/LacJ, SM/J, and C57BLKS/J inbred mice. We identified multiple significant QTL for red blood cell (RBC) count, hemoglobin (Hgb) and hematocrit (Hct) levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean cell hemoglobin concentration (CHCM). We identified four RBC count QTL: Rbcq1 (Chr 1, peak LOD score at 62 cM,), Rbcq2 (Chr 4, 60 cM), Rbcq3 (Chr 11, 34 cM), and Rbcq4 (Chr 10, 60 cM). Three MCV QTL were identified: Mcvq1 (Chr 7, 30 cM), Mvcq2 (Chr 11, 6 cM), and Mcvq3 (Chr 10, 60 cM). Single significant loci for Hgb (Hgbq1, Chr 16, 32 cM), Hct (Hctq1, Chr 3, 42 cM), and MCH (Mchq1, Chr 10, 60 cM) were identified. The data support the existence of a common RBC/MCH/MCV locus on Chr 10. Two QTL for CHCM (Chcmq1, Chr 2, 48 cM; Chcmq2, Chr 9, 44 cM) and an interaction between Chcmq2 with a locus on Chr 19 were identified. These analyses emphasize the genetic complexity underlying the regulation of erythroid peripheral blood traits in normal populations and suggest that genes not previously recognized as significantly impacting normal erythropoiesis exist.

Animals↗

PubMed2006

Mitoferrin is essential for erythroid iron assimilation.

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.

Anemia↗

PubMed2005

Role of hepatocyte nuclear factor 4alpha in control of blood coagulation factor gene expression.

Hepatocyte nuclear factor 4alpha (HNF4alpha) plays an important role in the maintenance of many liver-specific functions. Liver-specific HNF4alpha-null mice were used to determine whether hepatic HNF4alpha regulates blood coagulation in vivo. These mice exhibited reduced expression of hepatic coagulation factors V, IX, XI, XII, and XIIIB and a prolonged activated partial thromboplastin time but not prothrombin time. Promoter analysis of the mouse FXII and FXIIIB genes was performed to determine whether HNF4alpha directly regulates the genes encoding these coagulation factors. Sequence analysis revealed the presence of one and two HNF4alpha binding sites in the mouse FXII and FXIIIB genes, respectively. Using transient transfection and electrophoretic mobility shift analyses with the mouse FXII and FXIIIB promoters, it was established that the high levels of promoter activity were dependent on HNF4alpha binding sites and the expression of HNF4alpha. In conclusion, HNF4alpha has a critical role in blood coagulation homeostasis by directing transcription of the FXII and XIIIB genes.

Animals↗

PubMed2005

Quantitative trait loci for baseline white blood cell count, platelet count, and mean platelet volume.

A substantial genetic contribution to baseline peripheral blood counts has been established. We performed quantitative trait locus/loci (QTL) analyses to identify chromosome (Chr) regions harboring genes influencing the baseline white blood cell (WBC) count, platelet (Plt) count, and mean platelet volume (MPV) in F(2) intercrosses between NZW/LacJ, SM/J, and C57BLKS/J inbred mice. We identified six significant WBC QTL: Wbcq1 (peak LOD score at 38 cM, Chr 1), Wbcq2 (42 cM, Chr 3), Wbcq3 (0 cM, Chr 15), Wbcq4 (58 cM, Chr 1), Wbcq5 (82 cM, Chr 1), and Wbcq6 (8 cM, Chr 14). Three significant Plt QTL were identified: Pltq1 (24 cM, Chr 2), Pltq2 (36 cM, Chr 7), and Pltq3 (10 cM, Chr 12). Two significant MPV QTL were identified, Mpvq1 (62 cM, Chr 15) and Mpvq2 (44 cM, Chr 8). In total, the WBC QTL accounted for up to 31% of the total variance in baseline WBC count, while the Plt and MPV QTL accounted for up to 30% and 49% of the total variance, respectively. These analyses underscore the genetic complexity underlying these traits in normal populations and provide the basis for future studies to identify novel genes involved in the regulation of mammalian hematopoiesis.

Alleles↗

PubMed2005

Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis.

It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1-/-) exhibit moderate hemolytic anemia, with abnormal erythrocyte morphology (spherocytosis) and decreased membrane stability. Here, we characterized the Gardos channel function in vitro and in vivo in erythrocytes of 4.1-/- mice. Compared with wild-type, the Gardos channel of 4.1-/- erythrocytes showed an increase in Vmax (9.75 +/- 1.06 vs 6.08 +/- 0.09 mM cell x minute; P < .04) and a decrease in Km (1.01 +/- 0.06 vs 1.47 +/- 1.02 microM; P < .03), indicating an increased sensitivity to activation by intracellular calcium. In vivo function of the Gardos channel was assessed by the oral administration of clotrimazole, a well-characterized Gardos channel blocker. Clotrimazole treatment resulted in worsening of anemia and hemolysis, with decreased red cell survival and increased numbers of circulating hyperchromic spherocytes and microspherocytes. Clotrimazole induced similar changes in 4.2-/- and band 3+/- mice, indicating that these effects of the Gardos channel are shared in different models of murine spherocytosis. Thus, potassium and water loss through the Gardos channel may play an important protective role in compensating for the reduced surface-membrane area of hereditary spherocytosis (HS) erythrocytes and reducing hemolysis in erythrocytes with cytoskeletal impairments.

Anemia, Hemolytic↗

PubMed2005

N-myristoyltransferase 1 is essential in early mouse development.

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.

Acyltransferases↗

PubMed2005

Impaired synaptic plasticity and learning in mice lacking beta-adducin, an actin-regulating protein.

The adducin family of proteins interacts with the actin cytoskeleton and the plasma membrane in a calcium- and cAMP-dependent manner. Thus, adducins may be involved in changes in cytoskeletal organization resulting from synaptic stimulation. beta-Adducin knock-out mice were examined in physiological and behavioral paradigms related to synaptic plasticity to elucidate the role the adducin family plays in processes underlying learning and memory. In situ hybridization for alpha- and beta-adducin demonstrates that these mRNAs are found throughout the brain, with high levels of expression in the hippocampus. Schaffer collateral-CA1 tetanic long-term potentiation decayed rapidly in acute hippocampal slices from beta-adducin knock-out mice, although baseline spine morphology and postsynaptic density were normal. Interestingly, the input-output relationship was significantly increased in hippocampal slices from beta-adducin knock-out mice. Furthermore, beta-adducin knock-out mice were impaired in performance of fear conditioning and the water maze paradigm. The current results indicate that beta-adducin may play an important role in the cellular mechanisms underlying activity-dependent synaptic plasticity associated with learning and memory.

Actins↗

PubMed2004

Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex.

Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Adaptor Protein Complex 3↗

PubMed2004

Transgenic Cre expression mice for generation of erythroid-specific gene alterations.

Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.

Animals↗

PubMed2004

Implementing large-scale ENU mutagenesis screens in North America.

A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening.

Animals↗

PubMed2003

Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency.

Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model, wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygous wan mice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed in wan/wan mice from an F2 intercross between C3H/HeJ(+/wan) and CAST/Ei(+/+) F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2 wan homozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus, Hsm1 (hereditary spherocytosis modifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker for Spnb1 encoding erythroid beta-spectrin, an obvious candidate gene.

Animals↗

PubMed2003

Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene.

We mapped two new recessive mutations causing circling behavior and deafness to the same region on chromosome 7 and showed they are allelic by complementation analysis. One was named 'deaf circler' (allele symbol dfcr) and the other 'deaf circler 2 Jackson' (allele symbol dfcr-2J). Both were shown to be mutations of the Ush1c gene, the mouse ortholog of the gene responsible for human Usher syndrome type IC and for the non-syndromic deafness disorder DFNB18. The Ush1c gene contains 28 exons, 20 that are constitutive and eight that are alternatively spliced. The dfcr mutation is a 12.8 kb intragenic deletion that eliminates three constitutive and five alternatively spliced exons. The dfcr-2J mutation is a 1 bp deletion in an alternatively spliced exon that creates a transcriptional frame shift, changing 38 amino acid codons before introducing a premature stop codon. Both mutations cause congenital deafness and severe balance deficits due to inner ear dysfunction. The stereocilia of cochlear hair cells are disorganized and splayed in mutant mice, with subsequent degeneration of the hair cells and spiral ganglion cells. Harmonin, the protein encoded by Ush1c, has been shown to bind, by means of its PDZ-domains, with the products of other Usher syndrome genes, including Myo7a, Cdh23 and Sans. The complexes formed by these protein interactions are thought to be essential for maintaining the integrity of hair cell stereocilia. The Ush1c mutant mice described here provide a means to directly investigate these interactions in vivo and to evaluate gene structure-function relationships that affect inner ear and eye phenotypes.

Animals↗