Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells

2018 ◽  
Vol 156 (3) ◽  
pp. 150-157 ◽  
Author(s):  
Olga A. Efimova ◽  
Anna A. Pendina ◽  
Mikhail I. Krapivin ◽  
Vladimir V. Kopat ◽  
Andrei V. Tikhonov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Metaphase Plate ◽  
Embryonic Lung ◽  
Human Embryonic Lung ◽  
Accumulation Pattern ◽  
Chromosome Variability ◽  
Dna Strands ◽  
Cell To Cell Variability

5-hydroxymethylcytosine (5hmC) is an oxidative derivative of 5-methylcytosine (5mC). Recent studies have revealed a sharp difference in the levels of 5hmC in 2 opposite DNA strands of a given chromosome and a chromosome-wide cell-to-cell variability in mammalian cells. This asymmetric 5hmC distribution was found in cultured cells, which may not fully mimic in vivo epigenetic processes. We have checked whether inter-chromosome and inter-cell variability of 5hmC patterns is typical for noncultured human cells. Using indirect immunofluorescence, we analyzed the localization of 5hmC and its co-distribution with 5mC on direct preparations of mitotically active cells from human embryonic lung and chorionic cytotrophoblast samples. We demonstrated 3 types of chromosomes according to the 5hmC accumulation pattern: hydroxymethylated (5hmC in both sister chromatids), hemihydroxymethylated (5hmC in only 1 sister chromatid), and nonhydroxymethylated ones. Each accumulation type was not specific to any particular chromosome, resulting in different 5hmC patterns between homologous chromosomes, among chromosomes within each metaphase plate, among metaphases in one tissue, and between the tissues. The 5mC distribution was stable: chromosomes were methylated in R-bands and, especially in embryonic lung cells, in the heterochromatic regions 1q12, 9q12, and 16q11.2. Our results provide the first evidence of inter-cell and inter-chromosome variability of 5hmC patterns in human noncultured embryonic and extraembryonic cells.

scholarly journals Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent

2019 ◽  
Vol 47 (17) ◽  
pp. e97-e97 ◽  
Author(s):  
Kyle Meador ◽  
Christina L Wysoczynski ◽  
Aaron J Norris ◽  
Jason Aoto ◽  
Michael R Bruchas ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Functional Characterization ◽  
Cre Recombinase ◽  
Common Mechanism ◽  
Protein Fragments ◽  
Low Background ◽  
Wide Range ◽  
Split Protein

AbstractA common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

Immobilization and neutralization of Treponema pallidum attached to cultured mammalian cells

1985 ◽  
Vol 31 (12) ◽  
pp. 1152-1156
Author(s):  
Thomas Fitzgerald
Keyword(s):  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Cultured Cells ◽  
Treponema Pallidum ◽  
Immune Serum ◽  
Immune Reactivity ◽  
Complex Environment ◽  
In Vitro Effects

The in vitro effects of antibodies, complement, and (or) macrophages on Treponema pallidum have been previously characterized using relatively simple systems of organisms incubated with the immune components. In vivo, the more complex environment may alter immune reactivity. Experiments were performed to determine whether immobilizing and neutralizing antibodies retained their effectiveness in a more complex environment involving cultured mammalian cells. Two different protocols were used. In protocol A treponemes and normal or immune serum were mixed and added immediately to the cultured cells. In protocol B treponemes were preincubated for 18 h with cultured cells to maximize treponemal attachment; then normal or immune serum was added. With both protocols, attachment of organisms resulted in less effecient immobilization and neutralization. In further experiments, cultured cells were disrupted with Triton X, leaving cytoskeletal remnants on the vessel surface. Identical immobilization and neutralization experiments were performed in the presence of these remnants. In contrast to the findings with viable cultured cells, treponemal attachment to these nonviable remnants did not effect either antibody reaction. Attached organisms were immobilized or neutralized just as efficiently as unattached organisms. Results are discussed in terms of the altered immune reactivity in more complex in vitro environments.

scholarly journals Antimicrobial and Chemoattractant Activity, Lipopolysaccharide Neutralization, Cytotoxicity, and Inhibition by Serum of Analogs of Human Cathelicidin LL-37

2005 ◽  
Vol 49 (7) ◽  
pp. 2845-2850 ◽  
Author(s):  
Cristina D. Ciornei ◽  
Thorgerdur Sigurdardóttir ◽  
Artur Schmidtchen ◽  
Mikael Bodelsson
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Antimicrobial Properties ◽  
Neutrophil Granulocytes ◽  
Beneficial Effects ◽  
Hydrophobic Amino Acids ◽  
Conventional Antibiotics

ABSTRACT Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.

scholarly journals Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 161-171 ◽  
Author(s):  
S Mayrand ◽  
T Pederson
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Particle Structure ◽  
Nuclear Maturation ◽  
Shock Response ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
Drosophila Cells

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S.A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25 degrees C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25 degrees C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations.

scholarly journals Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2148-2154 ◽  
Author(s):  
R D McKinnon ◽  
P Danielson ◽  
M A Brow ◽  
F E Bloom ◽  
J G Sutcliffe
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Expression ◽  
Peripheral Tissues ◽  
Primate Cell

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

scholarly journals Membrane Insertion by Anthrax Protective Antigen in Cultured Cells

2005 ◽  
Vol 25 (13) ◽  
pp. 5492-5498 ◽  
Author(s):  
Maen Qa'dan ◽  
Kenneth A. Christensen ◽  
Lei Zhang ◽  
Thomas M. Roberts ◽  
R. John Collier
Keyword(s):  
Mammalian Cells ◽  
Time Course ◽  
Cultured Cells ◽  
Protective Antigen ◽  
Strong Increase ◽  
Membrane Insertion ◽  
Side Chains ◽  
Endosomal Membrane ◽  
Stirred Cell

ABSTRACT The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2β2-2β3 loops interact to generate a 14-strand transmembrane β-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2β2-2β3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the β-barrel model. Besides supporting the β-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo.

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2148-2154
Author(s):  
R D McKinnon ◽  
P Danielson ◽  
M A Brow ◽  
F E Bloom ◽  
J G Sutcliffe
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Expression ◽  
Peripheral Tissues ◽  
Primate Cell

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

scholarly journals DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Francesca Rossiello ◽  
Julio Aguado ◽  
Sara Sepe ◽  
Fabio Iannelli ◽  
Quan Nguyen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cultured Cells ◽  
Telomeric Dna ◽  
Cellular Events ◽  
Mouse Tissues ◽  
Damage Response ◽  
Dna Strands ◽  
Non Coding Rnas

Abstract The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

scholarly journals Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

2019 ◽  
Author(s):  
Ryan A. Flynn ◽  
Benjamin A. H. Smith ◽  
Alex G. Johnson ◽  
Kayvon Pedram ◽  
Benson M. George ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Cultured Cells ◽  
Mammalian Species ◽  
Cell Types ◽  
Intramolecular Interactions ◽  
Small Noncoding Rnas ◽  
Select Group ◽  
Domains Of Life

ABSTRACTGlycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells andin vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

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