Engineering Plant Alternative Oxidase Function in Mammalian Cells: Substitution of the Motif-like Sequence ENV for QDT Diminishes Catalytic Activity of Arum concinnatum AOX1a Expressed in HeLa Cells

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

Nature Communications ◽

10.1038/s41467-021-23892-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Praveen Bawankar ◽

Tina Lence ◽

Chiara Paolantoni ◽

Irmgard U. Haussmann ◽

Migle Kazlauskiene ◽

...

Keyword(s):

Catalytic Activity ◽

Sex Determination ◽

Ubiquitin Ligase ◽

Mammalian Cells ◽

E3 Ubiquitin Ligase ◽

Human Cells ◽

Biological Functions ◽

Mrna Metabolism ◽

Mrna Methylation ◽

Core Components

AbstractN6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

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Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active

Gene Therapy ◽

10.1038/s41434-021-00235-z ◽

2021 ◽

Author(s):

Luca Giordano ◽

Manish K. Aneja ◽

Natascha Sommer ◽

Nasim Alebrahimdehkordi ◽

Alireza Seraji ◽

...

Keyword(s):

Mammalian Cells ◽

Alternative Oxidase ◽

Chemically Modified ◽

Lung Carcinoma Cells ◽

Primary Mouse ◽

Catalytically Active ◽

Human Lung Carcinoma ◽

Pulmonary Arterial ◽

Metabolic Stresses ◽

Mitochondrial Respiratory

AbstractPlants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.

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Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin

Infection and Immunity ◽

10.1128/iai.70.9.4925-4935.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4925-4935 ◽

Cited By ~ 24

Author(s):

Spencer A. Leigh ◽

Kim S. Wise

Keyword(s):

Disulfide Bond ◽

Hela Cells ◽

Mammalian Cells ◽

Flow Cytometric Analysis ◽

Phase Variation ◽

Surface Proteins ◽

Mycoplasma Species ◽

Phase Variable ◽

Binding Region ◽

Mycoplasma Fermentans

ABSTRACT Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164-171.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

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Pathogenesis of shigella diarrhea. VII. Evidence for a cell membrane toxin receptor involving β1 {arrow} 4-linked N-acetyl-D-glucosamine oligomers

Journal of Experimental Medicine ◽

10.1084/jem.146.2.535 ◽

1977 ◽

Vol 146 (2) ◽

pp. 535-546 ◽

Cited By ~ 42

Author(s):

GT Keusch ◽

M Jacewicz

Keyword(s):

Cell Membrane ◽

Cholera Toxin ◽

Liver Cell ◽

Hela Cells ◽

Mammalian Cells ◽

Cell Membranes ◽

Proteolytic Enzymes ◽

Cell Monolayer ◽

Toxin Receptor ◽

Toxin Uptake

The binding of ShigeUa dysenteriae 1 cytotoxin to HeLa cells in culture and to isolated rat liver cell membranes was studied by means of an indirect consumption assay of toxicity from the medium, or by determination of cytotoxicity to the HeLa cell monolayer. Both liver cell membranes and HeLa cells removed toxicity from the medium during incubation, in contrast to WI-38 and Y-1 mouse adrenal tumor cells, both of which neither bound nor were affected by the toxin. Uptake of toxin was directly related to concentration of membranes added, time,and temperature, and indirectly related to the ionic strength of the buffer used. The chemical nature of the membrane receptor was characterized by using three principal approaches: (a) enzymatic sensitivity; (b) competitive inhibition and (c) receptor blockade studies. The receptor was destroyed by proteolytic enzymes, phospholipases (which markedly altered the gross appearance of the membrane preparation) and by lysozyme, but not by a variety of other enzymes. Of 28 carbohydrate and glycoprotein haptens studied, including cholera toxin and ganglioside, only the chitin oligosaccharide lysozyme substrates, per N-acetylated chitotriose, chitotetraose, and chitopentaose were effective competitive inhibitors. Greatest inhibition was found with the trimer, N, N', N" triacetyl chitotriose. Of three lectins studied as possible receptor blockers, including phytohemagglutinin, concanavalin A, and wheat germ agglutinin, only the latter, which is known to possess specific binding affinity for N, N', N" triacetyl chitotriose, was able to block toxin uptake. Evidence from all three approaches indicate, therefore, existence of a glycoprotein toxin receptor on mammalian cells, with involvement of oligomeric β1{arrow}4-1inked N-acetyl glucosamine in the receptor. This receptor is clearly distinct from the G(M1) ganglioside thought to be involved in the binding of cholera toxin to the cell membrane of a variety of cell types susceptible to its action.

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Association of Human DNA Topoisomerase IIα with Mitotic Chromosomes in Mammalian Cells Is Independent of Its Catalytic Activity

Experimental Cell Research ◽

10.1006/excr.1999.4616 ◽

1999 ◽

Vol 252 (1) ◽

pp. 50-62 ◽

Cited By ~ 31

Author(s):

Yin-Yuan Mo ◽

William T. Beck

Keyword(s):

Catalytic Activity ◽

Mammalian Cells ◽

Mitotic Chromosomes ◽

Topoisomerase Iiα ◽

Dna Topoisomerase ◽

Human Dna ◽

Dna Topoisomerase Iiα

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Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells

Molecules ◽

10.3390/molecules25194406 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4406

Author(s):

Maciej Studzian ◽

Paula Działak ◽

Łukasz Pułaski ◽

David M. Hedstrand ◽

Donald A. Tomalia ◽

...

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Fluorescence Lifetimes ◽

Intrinsic Luminescence ◽

Emission Properties ◽

Double Stranded Dna ◽

Cluster Type ◽

Transfection Agent

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.

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