scholarly journals Iron Superoxide Dismutases Targeted to the Glycosomes of Leishmania chagasi Are Important for Survival

2003 ◽  
Vol 71 (10) ◽  
pp. 5910-5920 ◽  
Author(s):  
Katherine A. Plewes ◽  
Stephen D. Barr ◽  
Lashitew Gedamu
Keyword(s):  
Fluorescent Protein ◽  
Parasite Growth ◽  
Leishmania Chagasi ◽  
Growth And Survival ◽  
Iron Superoxide Dismutase ◽  
Toxic Metabolites ◽  
Single Allele ◽  
Low Levels ◽  
Metabolic Activities ◽  
Green Fluorescent

ABSTRACT Kinetoplastid glycosomes contain a variety of metabolic activities, such as glycolysis, β-oxidation of fatty acids, lipid biosynthesis, and purine salvage. One advantage of sequestering metabolic activities is the avoidance of cellular oxidative damage by reactive oxygen species produced as a by-product of metabolism. Little is known about how glycosomes themselves withstand these toxic metabolites. We previously isolated an iron superoxide dismutase from Leishmania chagasi that is expressed at low levels in the early logarithmic promastigote stage and increases toward the stationary promastigote and amastigote stages. We have since identified a second highly homologous Lcfesodb gene that is expressed at high levels in the early logarithmic promastigote stage and decreases toward the stationary promastigote and amastigote stages. Localization studies using green fluorescent protein fusions have revealed that LcFeSODB1 and LcFeSODB2 are localized within the glycosomes by the last three amino acids of their carboxyl termini. To better understand the specific role that FeSODB plays in parasite growth and survival, a single-allele knockout of the Lcfesodb1 gene was generated. The parasites with these genes exhibited a significant reduction in growth when endogenous superoxide levels were increased with paraquat in culture. Furthermore, the FeSODB1-deficient parasites exhibited a significant reduction in survival within human macrophages. Our results suggest that LcFeSODB plays an important role in parasite growth and survival by protecting glycosomes from superoxide toxicity.

PU.1 determines the self-renewal capacity of erythroid progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3615-3623 ◽  
Author(s):  
Jonathan Back ◽  
Andrée Dierich ◽  
Corinne Bronn ◽  
Philippe Kastner ◽  
Susan Chan
Keyword(s):  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Self Renewal ◽  
Adult Mice ◽  
Erythroid Progenitor Cells ◽  
Low Levels ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

Abstract PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation—it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1-deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

Planta ◽  
2021 ◽  
Vol 254 (3) ◽  
Author(s):  
Bill Hendrix ◽  
Wei Zheng ◽  
Matthew J. Bauer ◽  
Ericka R. Havecker ◽  
Jennifer T. Mai ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Response To Treatment ◽  
Amaranthus Cruentus ◽  
Rnai Silencing ◽  
Endogenous Genes ◽  
Low Levels ◽  
Green Fluorescent ◽  
Rnai Response ◽  
Very High

Abstract Main conclusion 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. Abstract RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Regulation of the Arf tumor suppressor in Eμ-Myc transgenic mice: longitudinal study of Myc-induced lymphomagenesis

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 792-794 ◽  
Author(s):  
David Bertwistle ◽  
Charles J. Sherr
Keyword(s):  
Tumor Suppressor ◽  
Fluorescent Protein ◽  
Selective Pressure ◽  
Wild Type Allele ◽  
Wild Type ◽  
Coding Sequences ◽  
Cycle Arrest ◽  
Induced Tumors ◽  
Low Levels ◽  
Green Fluorescent

Abstract Lymphomagenesis in Eμ-Myc mice is opposed by the Arf tumor suppressor, whose inactivation compromises p53 function and accelerates disease. Finding nascent Eμ-Myc–induced tumors in which p19Arf causes cell-cycle arrest or apoptosis is problematic, since such cells will be eliminated until Arf or p53 function is lost. Knock-in mice expressing a green fluorescent protein (GFP) in lieu of Arf coding sequences allow analysis of Arfpromoter regulation uncoupled from p19Arf action. Prior to frank lymphoma development, unexpectedly low levels of Eμ-Myc–induced p19Arf or GFP were expressed. However, as lymphomas arose in Arf+/GFP heterozygotes, additional oncogenic events synergized with Eμ-Myc to further induce the functionally null Arf-Gfp allele. Concomitant up-regulation of p19Arf was not observed; instead, the wild-type allele was inactivated. We infer that very low levels of Arf are tumor suppressive, and that further induction provides the selective pressure for the emergence of tumors that have inactivated the gene.

Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells

2021 ◽  
Vol 118 (49) ◽  
pp. e2025748118
Author(s):  
Lei Wan ◽  
Xi Chen ◽  
Wolfhard Almers
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Membrane Lipids ◽  
Specific Interaction ◽  
Salt Bridges ◽  
Syntaxin 1A ◽  
Dense Cluster ◽  
Low Levels ◽  
Green Fluorescent

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

Growth and Survival of Attached Listeria on Lettuce and Stainless Steel Varies by Strain and Surface Type

2021 ◽  
Author(s):  
Lisa Gorski ◽  
Samarpita Walker ◽  
Kelly F Romanolo ◽  
Sophia Kathariou
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Foodborne Pathogen ◽  
Surface Growth ◽  
Growth And Survival ◽  
Abiotic Surfaces ◽  
Serotype 1 ◽  
Serotype 4B ◽  
Green Fluorescent

The foodborne pathogen Listeria monocytogenes lives as a saprophyte in nature and can adhere to and grows on surfaces as diverse as leaves, sediment, and stainless steel. To discern the mechanisms used by L. monocytogenes for attachment and growth on various surfaces, we studied interactions between the pathogen on lettuce and stainless steel. A panel of 24 strains (23 of Listeria monocytogenes and 1 L. innocua ) was screened for attachment and growth on lettuce at 4 o C and 25 o C and on stainless steel at 10 o C and 37 o C. Overnight growth of attached cells resulted in a 0 – 3 log increase on lettuce, depending on the strain and the temperature. Among the worst performing strains on lettuce were two from a large cantaloupe outbreak, indicating that factors important for interactions with cantaloupe may be different from those required on lettuce tissue. Strains that grew the best on lettuce belonged to serotypes 1/2a, 1/2b, and 4b and were from cheese, potatoes, and water/sediment near produce fields. Confocal microscopy of L. monocytogenes tagged with constitutively expressed green fluorescent protein indicated associations with the cut edges and veins of lettuce leaves. On stainless steel coupons, there was a 5 – 7 log increase at 10 o C after 7 d and a 4 – 7 log increase at 37 o C after 40 h. Statistically, surface growth on stainless steel was better for serotype 1/2a than for serotype 4b strains, even though certain serotype 4b strains grew well on the coupons. The latter included strains that originated from produce and water/sediment. Some strains were fit in both environments, while others showed variability between the two different surfaces. Further analysis of these strains should reveal molecular factors needed for adherence and surface growth of L. monocytogenes on different biotic and abiotic surfaces.

scholarly journals The three isoenzymes of human inositol-1,4,5-trisphosphate 3-kinase show specific intracellular localization but comparable Ca2+ responses on transfection in COS-7 cells

2003 ◽  
Vol 374 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Valérie DEWASTE ◽  
Colette MOREAU ◽  
Florence De SMEDT ◽  
Françoise BEX ◽  
Humbert De SMEDT ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Metabolizing Enzymes ◽  
Transfected Cells ◽  
Extracellular Signals ◽  
Inositol 1,4,5 Trisphosphate ◽  
Low Levels ◽  
Green Fluorescent ◽  
Mutant Cells

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64–66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343–351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400–405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529–535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 μM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals.

scholarly journals Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

2020 ◽  
Author(s):  
Bill Hendrix ◽  
Wei Zheng ◽  
Matthew J. Bauer ◽  
Ericka R. Havecker ◽  
Jennifer T. Mai ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Response To Treatment ◽  
Amaranthus Cruentus ◽  
Rnai Silencing ◽  
Secondary Sirnas ◽  
Endogenous Genes ◽  
Low Levels ◽  
Green Fluorescent ◽  
Rnai Response ◽  
Very High

Abstract22 nt miRNAs or siRNAs have been shown to specifically induce production of transitive (secondary) siRNAs for targeted mRNAs. An abrasion method to deliver dsRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for 3 endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GUN4. However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs, but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also had more transitive siRNAs produced in response to 22 nt siRNAs, but the response varied from weak (CHL-I) to strong (CHL-H). 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for GFP, CHL-H and GUN4 in N. benthamiana. The special activity of 22 nt siRNAs in producing a greater local phenotype and induction of elevated levels of transitive siRNAs was also shown in A. cruentus for the CHL-H gene. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Pathogenic role of Fgf23 in Hyp mice

2006 ◽  
Vol 291 (1) ◽  
pp. E38-E49 ◽  
Author(s):  
Shiguang Liu ◽  
Jianping Zhou ◽  
Wen Tang ◽  
Xi Jiang ◽  
David W. Rowe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Egfp Expression ◽  
Egfp Reporter ◽  
Low Levels ◽  
Green Fluorescent ◽  
Circulating Levels ◽  
Osteoblast Lineage ◽  
Inactivating Mutations

Inactivating mutations of the PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) endopeptidase, the disease-causing gene in X-linked hypophosphatemia (XLH), results in increased circulating levels of fibroblastic growth factor-23 (FGF23), a bone-derived phosphaturic factor. To determine the causal role of FGF23 in XLH, we generated a combined Fgf23-deficient enhanced green fluorescent protein (eGFP) reporter and Phex-deficient Hyp mouse model ( Fgf23+/−/ Hyp). eGFP expression was expressed in osteocytes embedded in bone that exhibited marked upregulation of eGFP in response to Phex deficiency and in CD31-positive cells in bone marrow venules that expressed low eGFP levels independently of Phex. In bone marrow stromal cells (BMSCs) derived from Fgf23−/−/ Hyp mice, eGFP expression was also selectively increased in osteocyte-like cells within mineralization nodules and detected in low levels in CD31-positive cells. Surprisingly, eGFP expression was not increased in cell surface osteoblasts, indicating that Phex deficiency is necessary but not sufficient for increased Fgf23 expression in the osteoblast lineage. Additional factors, associated with either osteocyte differentiation and/or extracellular matrix, are necessary for Phex deficiency to stimulate Fgf23 gene transcription in bone. Regardless, the deletion of Fgf23 from Hyp mice reversed the hypophosphatemia, abnormal 1,25(OH)2D3 levels, rickets, and osteomalacia associated with Phex deficiency. These results suggest that Fgf23 acts downstream of Phex to cause both the renal and bone phenotypes in Hyp mice.

scholarly journals Mutations in Caenorhabditis elegans actin, which are equivalent to human cardiomyopathy mutations, cause abnormal actin aggregation in nematode striated muscle

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 279 ◽  
Author(s):  
Yuriko Hayashi ◽  
Kanako Ono ◽  
Shoichiro Ono
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Central Component ◽  
C Elegans ◽  
Low Levels ◽  
Green Fluorescent ◽  
Cardiomyopathy Mutations

Actin is a central component of muscle contractile apparatuses, and a number of actin mutations cause diseases in skeletal, cardiac, and smooth muscles. However, many pathogenic actin mutations have not been characterized at cell biological and physiological levels. In this study, we tested whether the nematode Caenorhabditis elegans could be used to characterize properties of actin mutants in muscle cells in vivo. Two representative actin mutations, E99K and P164A, which cause hypertrophic cardiomyopathy in humans, are introduced in a muscle-specific C. elegans actin ACT-4 as E100K and P165A, respectively. When green fluorescent protein-tagged wild-type ACT-4 (GFP-ACT-4), is transgenically expressed in muscle at low levels as compared with endogenous actin, it is incorporated into sarcomeres without disturbing normal structures. GFP-ACT-4 variants with E100K and P165A are incorporated into sarcomeres, but also accumulated in abnormal aggregates, which have not been reported for equivalent actin mutations in previous studies. Muscle contractility, as determined by worm motility, is not apparently affected by expression of ACT-4 mutants. Our results suggest that C. elegans muscle is a useful model system to characterize abnormalities caused by actin mutations.

scholarly journals Various transgenic mouse lines to study proopiomelanocortin cells in the brain stem label disparate populations of GABAergic and glutamatergic neurons

2018 ◽  
Vol 315 (1) ◽  
pp. R144-R152 ◽  
Author(s):  
Andrew R. Rau ◽  
Alexander R. Hughes ◽  
Shane T. Hentges
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Glutamatergic Neurons ◽  
Pomc Mrna ◽  
Low Levels ◽  
Green Fluorescent ◽  
Transgenic Tools ◽  
The Brain ◽  
Mouse Lines ◽  
Transmitter Phenotype

Products of the proopiomelanocortin (POMC) prohormone regulate aspects of analgesia, reward, and energy balance; thus, the neurons that produce POMC in the hypothalamus have received considerable attention. However, there are also cells in the nucleus of the solitary tract (NTS) that transcribe Pomc, although low levels of Pomc mRNA and relative lack of POMC peptide products in the adult mouse NTS have hindered the study of these cells. Therefore, studies of NTS POMC cells have largely relied on transgenic mouse lines. Here, we set out to determine the amino acid (AA) transmitter phenotype of NTS POMC neurons by using Pomc-Gfp transgenic mice to identify POMC cells. We found that cells expressing the green fluorescent protein (GFP) represent a mix of GABAergic and glutamatergic cells as indicated by Gad2 and vesicular Glut2 ( vGlut2) mRNA expression, respectively. We then examined the AA phenotype of POMC cells labeled by a Pomc-Cre transgene and found that these are also a mix of GABAergic and glutamatergic cells. However, the NTS cells labeled by the Gfp- and Cre-containing transgenes represented distinct populations of cells in three different Pomc-Cre mouse lines. Consistent with previous work, we were unable to reliably detect Pomc mRNA in the NTS despite clear expression in the hypothalamus. Thus, it was not possible to determine which transgenic tool most accurately identifies NTS cells that may express Pomc or release POMC peptides, although the results indicate the transgenic tools for study of these NTS neurons can label disparate populations of cells with varied AA phenotypes.

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