scholarly journals Impairment in O-acetylserine-(thiol) lyase A and B, but not C, confers higher selenate sensitivity and uncovers role for A, B and C as L-Cys and L-SeCys desulfhydrases in Arabidopsis

2020 ◽  
Author(s):  
Assylay Kurmanbayeva ◽  
Aizat Bekturova ◽  
Aigerim Soltabayeva ◽  
Sudhakar Srivastava ◽  
Dinara Oshanova ◽  
...  
Keyword(s):  
Activity Levels ◽  
Wild Type ◽  
Catabolic Pathway ◽  
Total Glutathione ◽  
Starvation Response ◽  
Organic S ◽  
Protein Levels ◽  
Stress Symptoms ◽  
Cysteine Desulfhydrase

ABSTRACTThe role of the cytosolic O-acetylserine-(thiol) lyase A (OASTLA), chloroplastic OASTLB and mitochondrion OASTLC in plant resistance/sensitivity to selenate was studied in Arabidopsis plants. Impairment in OASTLA and B resulted in reduced biomass, chlorophyll and soluble protein levels compared with impaired OASTL C and Wild-Type treated with selenate. The lower organic-Se and protein-Se levels followed by decreased organic-S, S in proteins and total glutathione in oastlA and oastlB compared to Wild-Type and oastlC are indicative that Se accumulation is not the main cause for the stress symptoms, but rather the interference of Se with the S-reduction pathway. The increase in sulfite oxidase, adenosine 5′-phosphosulfate reductase, sulfite reductase and OASTL activity levels, followed by enhanced sulfite and sulfide, indicate a futile anabolic S-starvation response to selenate-induced organic-S catabolism in oastlA and oastlB compared to Wild-Type and oastlC.Additionally, the catabolic pathway of L-cysteine degradation was enhanced by selenate, and similar to L-cysteine producing activity, oastlA and B exhibited a significant decrease in L-cysteine desulfhydrase (DES) activity, compared with WT, indicating a major role of OASTLs in L-cysteine degradation. This notion was further evidenced by sulfide dependent DES in-gel activity, immunoblotting, immunoprecipitation with specific antibodies and identification of unique peptides in activity bands generated by OASTLA, B and C. Similar responses of the OASTLs in Seleno-Cysteine degradation was demonstrated in selenate stressed plants. Notably, no L-cysteine and L-Seleno-Cysteine DES activity bands but those related to OASTLs were evident. These results indicate the significance of OASTLs in degrading L-cysteine and L-SelenoCysteine in Arabidopsis.SummaryThe cytosolic OASTLA and chloroplastic OASTLB have significantly higher desulfhydrase activity rates than the cytosolic DES1 and are able to degrade L-Cys and L-SeCys to sulfide and selenide, respectively in Arabidopsis.

scholarly journals Interplay between Pds5 and Rec8 in regulating chromosome axis length and crossover frequency

2021 ◽  
Vol 7 (11) ◽  
pp. eabe7920
Author(s):  
Meihui Song ◽  
Binyuan Zhai ◽  
Xiao Yang ◽  
Taicong Tan ◽  
Ying Wang ◽  
...  
Keyword(s):  
Recombination Frequency ◽  
Crossover Frequency ◽  
Wild Type ◽  
Protein Levels ◽  
Homolog Pairing ◽  
Meiotic Chromosomes ◽  
Homologous Chromosome Pairing ◽  
Axis Length ◽  
Chromosome Axis

Meiotic chromosomes have a loop/axis architecture, with axis length determining crossover frequency. Meiosis-specific Pds5 depletion mutants have shorter chromosome axes and lower homologous chromosome pairing and recombination frequency. However, it is poorly understood how Pds5 coordinately regulates these processes. In this study, we show that only ~20% of wild-type level of Pds5 is required for homolog pairing and that higher levels of Pds5 dosage-dependently regulate axis length and crossover frequency. Moderate changes in Pds5 protein levels do not explicitly impair the basic recombination process. Further investigations show that Pds5 does not regulate chromosome axes by altering Rec8 abundance. Conversely, Rec8 regulates chromosome axis length by modulating Pds5. These findings highlight the important role of Pds5 in regulating meiosis and its relationship with Rec8 to regulate chromosome axis length and crossover frequency with implications for evolutionary adaptation.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Mayuri Tanaka-Yano ◽  
Dahai Wang ◽  
Eleanor Meader ◽  
Melissa A. Kinney ◽  
Vivian Morris ◽  
...  
Keyword(s):  
Young Adult ◽  
Developmental Stage ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Polycomb Group ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Developmental Maturation

Abstract Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis. First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7. Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels. As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold). To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors. Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

2019 ◽  
Author(s):  
Ewan K.S. McRae ◽  
Steven J. Dupas ◽  
Evan P. Booy ◽  
Ramanaguru S. Piragasam ◽  
Richard P. Fahlman ◽  
...  
Keyword(s):  
Mrna Levels ◽  
Wild Type ◽  
Biologically Relevant ◽  
Protein Levels ◽  
Guanine Quadruplex ◽  
Repressive Effect ◽  
G Quadruplex ◽  
Different Levels ◽  
In Cells

AbstractDDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we identified 26 proteins that are expressed at significantly different levels in cells expressing wild type DDX21 relative to an rG4 binding deficient DDX21 (M4). From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5’UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing only DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in cells expressing only DDX21 M4, and rendering previously resistant cells sensitive to TRAIL mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by a rG4 forming potential in their 5’UTRs.

Abstract 175: The Role of Axl in Accumulation of Immune Cells in Kidney and the Onset of Hypertension

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Sri Nagarjun Batchu ◽  
Angie Hughson ◽  
Janice Gerloff ◽  
Deborah J Fowell ◽  
Vyacheslav A Korshunov
Keyword(s):  
Blood Pressure ◽  
Immune Cells ◽  
Immune Cell ◽  
Kidney Injury ◽  
Wild Type ◽  
Protein Levels ◽  
Immune Cell Subsets ◽  
Early Increase ◽  
Salt Hypertension

Introduction: Gas6/Axl pathway contributes to elevation of blood pressure. Immune cells are implicated in initiation and maintenance of hypertension. In this study we aimed to investigate the role of Axl in immune cells on kidney injury and initiation of hypertension. Methods and Results: Deoxycorticosterone-acetate (DOCA; 75mg, 60days release) and salt hypertension was induced for 1wk or 6wks in four groups of Axl chimeras (n=4-5) that were generated by bone marrow (BM) transplant. Multi parameter flow cytometry was used to quantify five major immune cell subsets in digested kidneys from Axl chimeras. Systolic blood pressure (SBP) increased by 30mmHg in Axl+/+ →Axl+/+, Axl-/- →Axl-/- and Axl+/+ →Axl-/- mice after 1wk of DOCA-salt. However, chimeras that lack Axl in the BM cells (Axl-/- →Axl+/+) showed reduction in early increase in SBP (16+2mmHg). We observed a significant decrease in urine protein levels in Axl-/- →Axl+/+ (0.3+0.1μg/μl) compared to other Axl chimeras (∼0.7μg/μl) after 1wk of DOCA-salt. Kidney glomeruli areas were reduced in Axl-/- →Axl+/+ (4,143+229μm 2 ) compared to other Axl chimeras (∼6,000μm 2 ) after 6wks of DOCA-salt. Kidneys from Axl-/- →Axl-/- showed an increase in total leukocytes (8 vs. 4%), B cells (29 vs. 12%) and decrease in monocytes/macrophages (16 vs. 22%) and dendritic cells (5 vs. 10%) compared to Axl+/+ →Axl+/+. Moreover, Axl-/- →Axl+/+ showed further increase in leukocytes (17%), B (39%) and dendritic (13%) cells in kidneys compared to other Axl chimeras. In addition a small percentage of wild type T cells was increased in the kidneys from Axl-/- →Axl+/+ chimeras. Conclusions: These findings suggest that Axl expression in BM-derived cells is critical for kidney injury in DOCA-salt hypertension. Axl-dependent pathways regulate immune cell populations in the kidneys during initiation of hypertension. This study was supported by HL105623 grant (VAK)

Skeletal muscle reperfusion injury is enhanced in extracellular superoxide dismutase knockout mouse

2005 ◽  
Vol 289 (1) ◽  
pp. H181-H187 ◽  
Author(s):  
Jong Woong Park ◽  
Wen-Ning Qi ◽  
Yongting Cai ◽  
Igor Zelko ◽  
John Q. Liu ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Histological Analysis ◽  
Extracellular Superoxide Dismutase ◽  
Mrna Levels ◽  
Oxygen Species ◽  
Wild Type ◽  
Muscle Flaps ◽  
Protein Levels ◽  
Muscle Ischemia

This study investigates the role of extracellular SOD (EC-SOD), the major extracellular antioxidant enzyme, in skeletal muscle ischemia and reperfusion (I/R) injury. Pedicled cremaster muscle flaps from homozygous EC-SOD knockout (EC-SOD−/−) and wild-type (WT) mice were subjected to 4.5-h ischemia and 90-min reperfusion followed by functional and molecular analyses. Our results revealed that EC-SOD−/− mice showed significantly profound I/R injury compared with WT littermates. In particular, there was a delayed and incomplete recovery of arterial spasm and blood flow during reperfusion, and more severe acute inflammatory reaction and muscle damage were noted in EC-SOD−/− mice. After 90-min reperfusion, intracellular SOD [copper- and zinc-containing SOD (CuZn-SOD) and manganese-containing (Mn-SOD)] mRNA levels decreased similarly in both groups. EC-SOD mRNA levels increased in WT mice, whereas EC-SOD mRNA was undetectable, as expected, in EC-SOD−/− mice. In both groups of animals, CuZn-SOD protein levels decreased and Mn-SOD protein levels remained unchanged. EC-SOD protein levels decreased in WT mice. Histological analysis showed diffuse edema and inflammation around muscle fibers, which was more pronounced in EC-SOD−/− mice. In conclusion, our data suggest that EC-SOD plays an important role in the protection from skeletal muscle I/R injury caused by excessive generation of reactive oxygen species.

scholarly journals A role of the frontotemporal lobar degeneration risk factor TMEM106B in myelination

Brain ◽  
2020 ◽  
Vol 143 (7) ◽  
pp. 2255-2271 ◽  
Author(s):  
Tuancheng Feng ◽  
Rory R Sheng ◽  
Santiago Solé-Domènech ◽  
Mohammed Ullah ◽  
Xiaolai Zhou ◽  
...  
Keyword(s):  
Risk Factor ◽  
Frontotemporal Lobar Degeneration ◽  
Cathepsin D ◽  
Proteolipid Protein ◽  
Wild Type ◽  
Protein Levels ◽  
Lysosomal Protease ◽  
Precursor Cell Line ◽  
Lysosomal Membrane Protein

Abstract TMEM106B encodes a lysosomal membrane protein and was initially identified as a risk factor for frontotemporal lobar degeneration. Recently, a dominant D252N mutation in TMEM106B was shown to cause hypomyelinating leukodystrophy. However, how TMEM106B regulates myelination is still unclear. Here we show that TMEM106B is expressed and localized to the lysosome compartment in oligodendrocytes. TMEM106B deficiency in mice results in myelination defects with a significant reduction of protein levels of proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), the membrane proteins found in the myelin sheath. The levels of many lysosome proteins are significantly decreased in the TMEM106B-deficient Oli-neu oligodendroglial precursor cell line. TMEM106B physically interacts with the lysosomal protease cathepsin D and is required to maintain proper cathepsin D levels in oligodendrocytes. Furthermore, we found that TMEM106B deficiency results in lysosome clustering in the perinuclear region and a decrease in lysosome exocytosis and cell surface PLP levels. Moreover, we found that the D252N mutation abolished lysosome enlargement and lysosome acidification induced by wild-type TMEM106B overexpression. Instead, it stimulates lysosome clustering near the nucleus as seen in TMEM106B-deficient cells. Our results support that TMEM106B regulates myelination through modulation of lysosome function in oligodendrocytes.

scholarly journals A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion

2020 ◽  
Vol 95 (2) ◽  
pp. e01201-20
Author(s):  
Stacey Human ◽  
Anne L. Hotard ◽  
Christina A. Rostad ◽  
Sujin Lee ◽  
Louise McCormick ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Point Mutation ◽  
Stop Codon ◽  
Transcription Termination ◽  
Wild Type ◽  
Protein Levels ◽  
One Stop ◽  
Syncytial Virus ◽  
Tandem Stop Codons

ABSTRACTThis study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

scholarly journals Inhibition of fibrous adhesion formation in the temporomandibular joint of tenascin-C knockout mice

2014 ◽  
Author(s):  
Y. Shinohara ◽  
K. Okamoto ◽  
Y. Goh ◽  
N. Kiga ◽  
I. Tojyo ◽  
...  
Keyword(s):  
Wound Healing ◽  
Temporomandibular Joint ◽  
Smooth Muscle Actin ◽  
Adhesion Formation ◽  
Mouth Opening ◽  
Wild Type ◽  
Tenascin C ◽  
Protein Levels ◽  
Opening Method

Tenascin-C (TNC) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has been reported that TNC is expressed in inflamed synovial membranes and deformed discs of temporomandibular joint (TMJ) disorder. However, the role of TNC in TMJ is not fully known. In this study, the role of TNC in fibrous adhesion formation of TMJ was examined using TNC knockout (TNCKO) mice. Hypermobility was produced by excessive mouth opening method on the TMJ of both wild-type (WT) and TNCKO mice. TMJ wound healing was compared histologically, and the expression of TNC, fibronectin (FN) and α-smooth muscle actin (α-SMA) in the wounded TMJ was examined by immunohistochemical and immunoblot analyses. Based on histologic analysis, fibrous adhesions were observed in the TMJ of both TNCKO and wild-type (WT) mice after excessive mouth opening. However, fibrous adhesion formation in TNCKO mice occurred later than in WT mice. TNC was expressed in the wounded TMJ disc and mandibular fossa. Although FN and α-SMA expression in the TMJ of TNCKO and WT mice was up-regulated after excessive mouth opening, FN and α-SMA protein levels were higher in WT mice at the same time points. In the wounded TMJ, TNC appears to enhance the expression of FN and α-SMA, and a lack of TNC may reduce fibrous adhesion formation in the TMJ. TNC plays an important role in TMJ wound healing, especially for wounds generated by mechanical stress.

Abstract 345: Effects Of Endothelial-targeted Overexpression Of Nox4 On Vascular Tone And Blood Pressure In Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Robin Ray ◽  
Min Zhang ◽  
Alison C Brewer ◽  
Ajay M Shah
Keyword(s):  
Blood Pressure ◽  
Transgenic Mice ◽  
Fold Increase ◽  
Wild Type ◽  
Protein Levels ◽  
Promoter Construct ◽  
Activation Mechanisms ◽  
Endothelium Dependent Relaxation

NADPH oxidases (Noxs) are major sources of reactive oxygen species (ROS) that are involved in the pathophysiology of several cardiovascular disorders. Of the 5 Nox isoforms identified to date, Nox2 and Nox4 are the main isoforms expressed in the endothelium. Whereas Nox2 has been implicated in the genesis of endothelial dysfunction, the role of Nox4 remains unclear. Interestingly, the activation mechanisms of Nox2 and Nox4 appear to be distinct. To specifically examine the function of endothelial Nox4 in vivo , we generated transgenic mice with endothelial-targeted overexpression of Nox4 using a Tie2 promoter construct. Nox4 transgenic mice (TG) backcrossed onto a C57BL/6J background had increased Nox4 mRNA in endothelial-rich tissues and in isolated coronary microvascular endothelial cells (CMEC) compared to wild-type littermates (WT) (2-fold increase in CMEC; p<0.001). Aortic Nox4 protein levels were 3-fold higher in TG compared to WT. CMEC isolated from TG mice had increased NADPH-dependent superoxide production compared to WT (237.6 ± 2.7 vs. 186.5 ± 7.1 integrated RLU; n = 3, p<0.01) as well as increased H 2 O 2 production (7.60 ± 0.70 vs. 3.22 ± 0.42 μM H 2 O 2 /105 cells; n=3, p<0.01). No changes were detected in mRNA expression of SOD1, SOD2, SOD3, catalase or eNOS in aorta of TG compared to WT mice. Isolated aortic rings from TG mice exhibited enhanced endothelial-dependent vasorelaxation to cumulative addition of acetylcholine compared to WT (−log EC 50 7.76 ± 0.07 vs. 7.20 ± 0.05; n =12, p<0.001), a difference that was abolished by catalase (1500 units/ml). There was no difference in endothelial-independent responses to sodium nitroprusside (−log EC 50 8.57 ± 0.11 vs. 8.54 ± 0.09; n = 12, p = NS). In vivo blood pressure measured both by tail-cuff plethysmography and ambulatory telemetry was significantly lower in TG compared to WT (systolic 117.4 ± 1.9 vs. 125.5 ± 2.1 mmHg and diastolic 90.1 ± 2.0 vs. 98.1 ± 2.1 mmHg by telemetry; n =5, p<0.05). These results indicate that modest endothelium-targeted overexpression of Nox4 in vivo enhances endothelium-dependent relaxation and reduces blood pressure, probably through increased generation of H 2 O 2 . These in vivo effects are quite distinct from those that have been found with Nox2 overexpression.

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