scholarly journals Human mutation D157G in ferroportin leads to hepcidin-independent binding of Jak2 and ferroportin down-regulation

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2956-2959 ◽  
Author(s):  
Ivana De Domenico ◽  
Eric Lo ◽  
Diane M. Ward ◽  
Jerry Kaplan
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Transferrin Saturation ◽  
High Serum ◽  
Growth Defect ◽  
Down Regulation ◽  
Severe Growth Defect ◽  
Human Mutation ◽  
Green Fluorescent ◽  
Severe Growth

Abstract Mutations in the iron exporter ferroportin (Fpn) result in iron overload in macrophages or hepatocytes depending upon the mutation. Patients with Fpn mutation D157G show high serum ferritin and normal to slightly elevated transferrin saturation. Here, we show that Fpn(D157G)–green fluorescent protein (GFP) is down-regulated independent of hepcidin, and that this down-regulation is due to the constitutive binding of Jak2 and Fpn phosphorylation. Expression of Fpn(D157G)-GFP in Danio rerio results in a severe growth defect, which can be rescued by iron supplementation. These results identify a hepcidin-independent regulation of Fpn that can result in alterations in iron homeostasis.

scholarly journals Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA

2005 ◽  
Vol 25 (11) ◽  
pp. 4359-4370 ◽  
Author(s):  
Suresh K. Purushothaman ◽  
Janusz M. Bujnicki ◽  
Henri Grosjean ◽  
Bruno Lapeyre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Zinc Binding ◽  
Growth Defect ◽  
Putative Protein ◽  
Yeast Trna ◽  
Protein Methyltransferase ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT N 2 -Monomethylguanosine-10 (m2G10) and N 2 ,N 2 -dimethylguanosine-26 (m2 2G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m2 2G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m2 2G26 affects tRNA metabolism or functioning.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals Functional properties of human ferroportin, a cellular iron exporter reactive also with cobalt and zinc

2014 ◽  
Vol 306 (5) ◽  
pp. C450-C459 ◽  
Author(s):  
Colin J. Mitchell ◽  
Ali Shawki ◽  
Tomas Ganz ◽  
Elizabeta Nemeth ◽  
Bryan Mackenzie
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Serum Zinc ◽  
Functional Expression ◽  
Extracellular Ph ◽  
Zinc Homeostasis ◽  
Affinity Constant ◽  
Protein Fusion ◽  
Zinc Levels ◽  
Green Fluorescent

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with 55Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing 55Fe efflux up to 300-fold over control. Ferroportin-mediated 55Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10−7 M. Ferroportin expression also stimulated efflux of 65Zn and 57Co but not of 64Cu, 109Cd, or 54Mn. Hepcidin treatment of oocytes inhibited efflux of 55Fe, 65Zn, and 57Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.

scholarly journals bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

PLoS ONE ◽  
2017 ◽  
Vol 12 (6) ◽  
pp. e0179513 ◽  
Author(s):  
Ritika Kar ◽  
Prachi Nangpal ◽  
Shubhita Mathur ◽  
Swati Singh ◽  
Anil K. Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pigs ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

2017 ◽  
Author(s):  
Andrian Gutu ◽  
Frederick Chang ◽  
Erin K. O‘Shea
Keyword(s):  
Spatial Distribution ◽  
Thylakoid Membrane ◽  
Growth Conditions ◽  
Dynamical Localization ◽  
Growth Defect ◽  
Cell Periphery ◽  
Diffuse Fraction ◽  
Photosynthetic Complexes ◽  
Severe Growth Defect ◽  
Severe Growth

SUMMARYVipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery

Nanomedicine ◽  
2021 ◽  
Author(s):  
Akbar Hasanzadeh ◽  
Fatemeh Radmanesh ◽  
Elaheh Sadat Hosseini ◽  
Iman Hashemzadeh ◽  
Jafar Kiani ◽  
...  
Keyword(s):  
Carbon Dots ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
High Serum ◽  
Spectroscopic Techniques ◽  
Delivery Vector ◽  
One Step ◽  
Diphenyl Tetrazolium Bromide ◽  
Green Fluorescent

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

scholarly journals Insufficient levels of thenrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. colistrain

2017 ◽  
Vol 104 (3) ◽  
pp. 377-399 ◽  
Author(s):  
Vignesh M. P. Babu ◽  
Mark Itsko ◽  
Jamie C. Baxter ◽  
Roel M. Schaaper ◽  
Mark D. Sutton
Keyword(s):  
Ribonucleotide Reductase ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals RelA/p65 Regulation of IκBβ

2005 ◽  
Vol 25 (12) ◽  
pp. 4956-4968 ◽  
Author(s):  
Erin Hertlein ◽  
Jingxin Wang ◽  
Katherine J. Ladner ◽  
Nadine Bakkar ◽  
Denis C. Guttridge
Keyword(s):  
Postnatal Development ◽  
26S Proteasome ◽  
Carboxyl Terminus ◽  
Growth Defect ◽  
Tight Control ◽  
Resistant Form ◽  
Specific Manner ◽  
Severe Growth Defect ◽  
Severe Growth ◽  
Hyperphosphorylated Form

ABSTRACT IκB inhibitor proteins are the primary regulators of NF-κB. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known regarding the regulation of IκBβ by NF-κB. Here, we describe in detail the regulation of IκBβ by RelA/p65. Using p65 −/− fibroblasts, we show that IκBβ is profoundly reduced in these cells, but not in other NF-κB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65 −/− cells and tissues, IκBα is also reduced, but not nearly to the same extent as IκBβ, thus highlighting the degree to which IκBβ is dependent on p65. This dependence is based on the ability of p65 to stabilize IκBβ protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IκBβ was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65 −/− fibroblasts, expression of a proteolysis-resistant form of IκBβ, but not IκBα, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IκBβ protein by p65 is necessary for the maintenance of cellular homeostasis.

scholarly journals Up-regulation of HTR1A reverses stress-induced visceral hypersensitivity through modulating interactions among the anterior cingulate cortex, insular cortex and hippocampus

Pteridines ◽  
2020 ◽  
Vol 31 (1) ◽  
pp. 165-173
Author(s):  
Liqian Xuan ◽  
Jingjing Zhou ◽  
Lisha Yi ◽  
Shuchang Xu
Keyword(s):  
Anterior Cingulate Cortex ◽  
Psychological Stress ◽  
Opposite Effect ◽  
Fluorescent Protein ◽  
Insular Cortex ◽  
Cingulate Cortex ◽  
Visceral Hypersensitivity ◽  
Anterior Cingulate ◽  
Down Regulation ◽  
Green Fluorescent

AbstractBackground: This study aimed to explore the effect of 5-HT1A receptors (HTR1A) on activation of the anterior cingulate cortex and simultaneous regulation of neural activity in the insular cortex and hippocampus.Methods: The IBS rat model was established via chronic water avoidance stress (WAS). Visceral sensitivity was measured by electromyogram, and anxiety-like behaviours were evaluated by the open field test. HTR1A-specific lentivirus expressing green fluorescent protein was used to overexpress or down-regulate HTR1A expression. Protein expression levels were detected by western blot.Results: Up-regulation of HTR1A in ACC could inhibit ACC sensitization and reverse the visceral hypersensitivity and anxiety-like behaviours induced by chronic psychological stress. In contrast, down-regulation of HTR1A in ACC might promote these behaviors in IBS rats. Additionally, up-regulation of HTR1A in ACC could inhibit IC and hippocampus sensitization, while down-regulation might have the opposite effect.Conclusions: In IBS rats, HTR1A could modulate ACC activation and interactions among the ACC, IC and hippocampus. These effects might in turn contribute to the development of visceral hypersensitivity and anxiety-like behaviours induced by chronic psychological stress.

scholarly journals The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

2021 ◽  
Author(s):  
Majeed Bakari-Soale ◽  
Nonso Josephat Ikenge ◽  
Marion Scheibe ◽  
Falk Butter ◽  
Nicola Gail Jones ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Growth Defect ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Comprehensive Picture ◽  
Severe Growth Defect ◽  
H Protein ◽  
Severe Growth

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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