Expression of Tropomodulin1 (Tmod1) in the Heart Rescues Embryonic Lethality of Tmod1 Null Mice and Results in a Mild Hemolytic Anemia Due to Absence of Tmod1 in Red Blood Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 807-807
Author(s):  
Velia M. Fowler ◽  
Jeannette Moyer ◽  
Roberta Nowak ◽  
Kimberly Fritz-Six ◽  
Carmela Ferreira-Mota ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Western Blotting ◽  
Actin Filaments ◽  
Membrane Skeleton ◽  
Yolk Sac ◽  
Embryonic Lethality ◽  
Mean Corpuscular Hemoglobin ◽  
Null Mouse ◽  
Distribution Width ◽  
Rbc Membrane

Abstract Tropomodulin1 (Tmod1) caps the pointed ends of actin filaments in the red blood cell (RBC) membrane skeleton and is proposed to regulate actin filament lengths and organization in the membrane skeleton. Tmod1 is first expressed at E7.5 of mouse embryogenesis in blood islands of the yolk sac and in the cardiac myocytes of the developing heart tube. Targeted deletion of the Tmod1 gene in mice leads to abnormal cardiac development and defective vasculogenesis and hematopoesis in the yolk sac, followed by embryonic lethality between E9.5–10. To investigate the function of Tmod1 in RBCs we rescued the embryonic lethality of the Tmod1 null mouse by crossing with a TOT mouse that expresses a Tmod1 transgene in the heart under the control of the α-myosin heavy chain promoter. Genotyping of litters from crosses of Tmod1-/+;TOT+ mice with Tmod1+/− mice demonstrates that embryos with no endogenous Tmod1 but expressing the Tmod1 transgene (Tmod1−/−;TOT+) develop to term with no apparent defects in vasculogenesis or hematopoeisis. Western blotting and immunofluorescence staining demonstrates that Tmod1 protein is present in the heart but absent from RBCs in Tmod1−/−;TOT+ rescued embryos or adult mice. Instead, the Tmod3 isoform that is normally present in embryonic RBCs, but not detected in adult RBCs of wild-type mice, is increased on the membranes of Tmod1-deficient adult RBCs. Hematological analyses reveal that these Tmod1-deficient mice exhibit a compensated mild hemolytic anemia, with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), increased red cell distribution width (RDW), and reticulocytosis. Quantitative western blotting demonstrates that major RBC membrane proteins are not altered in the absence of Tmod1, but that there is about a two-fold increase in levels of actin and tropomyosin on the membrane, suggesting that actin filaments may be longer than in normal RBCs. We conclude that 1) Tmod1 in RBCs is not required for embryonic development or viability, 2) yolk sac and hematopoetic defects in Tmod1 null mice are secondary to cardiac defects, 3) Tmod’s function in embryonic RBCs may be performed by the Tmod3 isoform that is normally expressed in these RBCs, and 4) the absence of Tmod1 in adult RBCs is incompletely compensated by up-regulation of Tmod3, leading to altered actin filaments, decreased membrane stability and a mild hemolytic anemia.

Targeted Deletion of γ-Adducin in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1723-1723
Author(s):  
Kenneth E. Sahr ◽  
Amy J. Lambert ◽  
Steven L. Ciciotte ◽  
Luanne L. Peters
Keyword(s):  
Western Blotting ◽  
Peripheral Blood ◽  
Osmotic Fragility ◽  
Membrane Skeleton ◽  
Red Cells ◽  
Null Mouse ◽  
Exon 2 ◽  
Rbc Membrane ◽  
Blood Smears ◽  
Peripheral Blood Smears

Abstract The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (<5% of normal). These studies show that (1) loss of γ-adducin alone does not significantly impact RBC membrane skeleton structure and function; (2) α- and β-adducin are stable and present at normal levels in the absence of γ-adducin; (3) the loss of γ-adducin in β-adducin null mice does not further exacerbate the β-adducin null HS phenotype; (4) the exacerbated loss of α-adducin in βγ double null RBCs suggests that up-regulated γ-adducin in β-adducin null mice associates in some way with and stabilizes α-adducin in the RBC membrane, but is unable to compensate functionally for the loss of the β subunit. We conclude that the normal function and stable incorporation of adducin into the peripheral membrane skeleton of red cells requires the presence of heterologous αβ binding subunits. Additional studies of adducin null mouse models, including our recently generated α-adducin null strain, will be useful tools in defining adducin functions and interactions in multiple tissues and organs.

Targeted Deletion of α-Adducin Results in Absent β-Adducin, Compensated Hemolytic Anemia, and Hydrocephalus in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 141-141 ◽  
Author(s):  
Raymond F. Robledo ◽  
Steven L. Ciciotte ◽  
Babette Gwynn ◽  
Kenneth E. Sahr ◽  
Diana M. Gilligan ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Western Blotting ◽  
Systemic Blood Pressure ◽  
Membrane Skeleton ◽  
Postnatal Growth ◽  
Null Mouse ◽  
Erythroid Cells ◽  
Targeted Deletion ◽  
Protein Functions ◽  
The Brain

Abstract The membrane skeleton underlies the lipid bilayer and imparts strength and deformability to the red blood cell (RBC). Spectrin, the major component of the membrane skeleton, is crosslinked by short actin filaments into a two-dimensional array at junctional complexes, which are composed of multiple additional proteins including adducin. Three mammalian adducins (α, β, γ) exist that are encoded by distinct genes (Add1, Add2, Add3). α- and γ-adducin are ubiquitously expressed, with the highest expression of α-adducin seen in erythroid cells, brain, kidney, and heart. Expression of β-adducin is restricted to erythroid cells and brain. In RBCs heterotetramers of α- and β-adducin regulate actin filament length via barbed end capping. Previously, we deleted β-adducin in mice. Loss of β-adducin resulted in decreased α-adducin and up-regulated γ-adducin (5-fold), producing fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Here, we report on recently generated α-adducin null mice. We inactivated the α-adducin gene (Add1) by targeted deletion of exons 10–12, and confirmed by western blotting the complete loss of α-adducin in RBC ghosts and in the brain. All mice were maintained on a segregating B6.129 genetic background. Heterozygous mice are normal in all parameters examined to date. Notably, although β-adducin gene expression is normal, western blotting revealed a complete absence of β-adducin protein in α-adducin null RBC ghosts. γ-adducin, present at low levels in normal mouse RBCs, was unchanged, as were all other membrane skeleton proteins examined by SDS-PAGE and/or western blotting (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). α-adducin null mice display characteristics of mild compensated hemolytic anemia. The hematocrit is significantly decreased (43 vs. 46% in wildtype). RBCs are microcytic (MCV 41.9 vs. 46.4 fL) and osmotically fragile (50% lysis at 215 mOsm NaCl vs. 190 mOsm). The MCH is significantly decreased while the MCHC is significantly elevated, suggestive of RBC dehydration. The percentage of circulating reticulocytes is significantly increased (5.0 vs. 2.7%). Spleen weights are normal. Examination of peripheral blood smears and scanning electron microscopy confirms microcytic, anisocytotic RBCs with spherocytes and elliptocytes present. α-adducin null mice show postnatal growth retardation and approximately 66% develop critical hydrocephalus with marked expansion of the lateral and third ventricles by the fourth month of age. Normal littermates never show hydrocephalus. We conclude: α-adducin null mice have mild, compensated hemolytic anemia; β-adducin is unstable in RBCs in the absence of α-adducin; loss of α-adducin in the brain is associated with a high incidence of lethal hydrocephalus. Additional studies of the α-adducin null mouse model will be useful in defining protein functions and interactions in RBCs, the requirement for adducin in platelet function, the role of adducin in the brain, and its role in the regulation of systemic blood pressure.

Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of α-Spectrin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1662-1662
Author(s):  
Raymond F. Robledo ◽  
Amy J. Lambert ◽  
Babette Gwynn ◽  
Lucy B. Rowe ◽  
Diana M. Gilligan ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Chromosome 1 ◽  
Major Protein ◽  
Blood Profile ◽  
Rbc Membrane ◽  
C Terminus ◽  
Sds Page ◽  
Rbc Membrane Skeleton

Abstract The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

scholarly journals Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2590-2599 ◽  
Author(s):  
Jeannette D. Moyer ◽  
Roberta B. Nowak ◽  
Nancy E. Kim ◽  
Sandra K. Larkin ◽  
Luanne L. Peters ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Filament Length ◽  
Western Blots ◽  
Rbc Membrane ◽  
Cellular Dehydration ◽  
Rbc Membrane Skeleton

Abstract The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

scholarly journals Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

2015 ◽  
Vol 26 (9) ◽  
pp. 1699-1710 ◽  
Author(s):  
David S. Gokhin ◽  
Roberta B. Nowak ◽  
Joseph A. Khoory ◽  
Alfonso de la Piedra ◽  
Ionita C. Ghiran ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Microfluidic Channel ◽  
Biomechanical Properties ◽  
Membrane Skeleton ◽  
Single Filament ◽  
Rbc Membrane ◽  
Filament Assembly ◽  
Membrane Oscillations ◽  
Resealed Ghosts

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.

Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1728-1728
Author(s):  
Jason M. Wooden ◽  
Greg L. Finney ◽  
Michael J. MacCoss ◽  
Luanne L. Peters ◽  
Diana M. Gilligan
Keyword(s):  
Mouse Models ◽  
Hemolytic Anemia ◽  
Membrane Skeleton ◽  
Tandem Mass ◽  
Secondary Effects ◽  
Knock Out ◽  
Rbc Membrane ◽  
Protein 4.2 ◽  
Knock Out Mice ◽  
Rbc Membrane Skeleton

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up’ analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.

E-Tmod capping of actin filaments at the slow-growing end is required to establish mouse embryonic circulation

2003 ◽  
Vol 284 (5) ◽  
pp. H1827-H1838 ◽  
Author(s):  
Xin Chu ◽  
Ju Chen ◽  
Mary C. Reedy ◽  
Carlos Vera ◽  
K.-L. Paul Sung ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cardiac Contractility ◽  
Embryonic Stem ◽  
Membrane Skeleton ◽  
Yolk Sac ◽  
Striated Muscles ◽  
Erythroid Cells ◽  
Heart Tube ◽  
The Right ◽  
Slow Growing

Tropomodulins are a family of proteins that cap the slow-growing end of actin filaments. Erythrocyte tropomodulin (E-Tmod) stabilizes short actin protofilaments in erythrocytes and caps longer sarcomeric actin filaments in striated muscles. We report the knockin of the β-galactosidase gene ( LacZ) under the control of the endogenous E-Tmodpromoter and the knockout of E-Tmod in mouse embryonic stem cells. E-Tmod−/−embryos die around embryonic day 10 and exhibit a noncontractile heart tube with disorganized myofibrils and underdevelopment of the right ventricle, accumulation of mechanically weakened primitive erythroid cells in the yolk sac, and failure of primary capillary plexuses to remodel into vitelline vessels, all required to establish blood circulation between the yolk sac and the embryo proper. We propose a hemodynamic “plexus channel selection” mechanism as the basis for vitelline vascular remodeling. The defects in cardiac contractility, vitelline circulation, and hematopoiesis reflect an essential role for E-Tmod capping of the actin filaments in both assembly of cardiac sarcomeres and of the membrane skeleton in erythroid cells that is not compensated for by other proteins.

scholarly journals Impacts of Mycoplasma loads and lung lesions on immune and hematological statuses of pigs in an eight-breed cross heterogeneous population

2020 ◽  
Vol 98 (8) ◽  
Author(s):  
Qing Zhang ◽  
Jing Li ◽  
Tao Huang ◽  
Yifeng Zhang ◽  
Wenwu Xu ◽  
...  
Keyword(s):  
Blood Cell ◽  
Neutrophil Count ◽  
Lymphocyte Count ◽  
Hemoglobin Concentration ◽  
Mean Corpuscular Hemoglobin ◽  
Volume Distribution ◽  
Mean Corpuscular Hemoglobin Concentration ◽  
Corpuscular Hemoglobin ◽  
Distribution Width ◽  
Lung Lesions

Abstract Developments of pulmonary diseases, often accompanied by infections of bacteria, severely affect the meat production and welfare of pigs. This study investigated 307 pigs at age of 240 d from an eight-breed cross reared under standardized housing conditions for associations among the extent of lung lesions, bacteria load inferred from 16S rRNA sequencing of bronchoalveolar lavage fluid, as well as 57 immune cells and 25 hematological traits. We showed that the pigs under study suffered substantial and varied lung lesions, and the Mycoplasma is the most associated bacteria genera. At a false discovery rate of 0.05 (FDR < 0.05), the severity of lung lesions were significantly associated with greater CD8+ to CD3+ cell ratio, neutrophil-to-lymphocyte ratio (NLR), and standard deviation of red blood cell volume distribution width (RDW-SD), and lower CD4−CD8−/CD3+, CD3+CD4−CD8−/PBMCs (peripheral blood mononuclear cells) and CD14−CD16−/PBMCs cell ratios, mean corpuscular hemoglobin concentration, lymphocyte count, and lymphocyte count percentage, reflecting an status of inflammation, immune suppression, and hypoxia of the pigs accompanying the progression of the lung lesions. The Mycoplasma abundance showed positive correlations with neutrophil count, neutrophil count percentage, NLR, monocyte count, coefficient of variation in red blood cell volume distribution width , and RDW-SD, and negative correlations with mean corpuscular hemoglobin concentration, lymphocyte count, and lymphocyte count percentage; these correlations are largely consistent with those of lung lesions, supporting the comorbidity of lung lesions and Mycoplasma infection. We also observed nonlinear associations that sharp increases in neutrophil count and neutrophil count percentage occurred only when Mycoplasma abundance raised above the population-average level. The results provide helpful insights into the changes of host immune status in response to Mycoplasma relevant lung diseases in pigs.

scholarly journals The Role of Mcl-1 in Embryonic Neural Precursor Cell Apoptosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Robert T. Flemmer ◽  
Sarah P. Connolly ◽  
Brittany A. Geizer ◽  
Joseph T. Opferman ◽  
Jacqueline L. Vanderluit
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Precursor Cell ◽  
Embryonic Lethality ◽  
Conditional Knockout ◽  
Neural Precursor ◽  
Null Mouse ◽  
Neural Precursor Cell ◽  
Thoracic Spinal Cord

Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, regulates neural precursor cell (NPC) survival in both the developing and adult mammalian nervous system. It is unclear when during the neurogenic period Mcl-1 becomes necessary for NPC survival and whether Bax is the sole pro-apoptotic target of Mcl-1. To address these questions, we used the nervous system-specific Nestin-Cre Mcl-1 conditional knockout mouse line (Mcl-1 CKO) to assess the anti-apoptotic role of Mcl-1 in developmental neurogenesis. Loss of Mcl-1 resulted in a wave of apoptosis beginning in the brainstem and cervical spinal cord at embryonic day 9.5 (E9.5) and in the forebrain at E10.5. Apoptosis was first observed ventrally in each region and spread dorsally over time. Within the spinal cord, apoptosis also spread in a rostral to caudal direction following the path of differentiation. Breeding the Mcl-1 CKO mouse with the Bax null mouse rescued the majority of NPC from apoptosis except in the dorsomedial brainstem and ventral thoracic spinal cord where only 50% were rescued. This demonstrates that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis. Interestingly, although co-deletion of Bax rescued the majority of NPC apoptosis, it resulted in embryonic lethality at E13, whereas conditional deletion of both Mcl-1 and Bax rescued embryonic lethality. In summary, this study demonstrates the widespread dependency on Mcl-1 during nervous system development.

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