scholarly journals The “adductome”: a limited repertoire of adducted proteins in human cells

2019 ◽  
Author(s):  
Kostantin Kiianitsa ◽  
Nancy Maizels
Keyword(s):  
Nucleic Acids ◽  
Rna Splicing ◽  
Genomic Structure ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Cells ◽  
Hmg Proteins ◽  
Proliferating Cells ◽  
Proteomic Approach ◽  
Low Concentrations

ABSTRACTProteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that “RADAR-MS” can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r=0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins — the “adductome” — is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes

1996 ◽  
Vol 319 (3) ◽  
pp. 887-896 ◽  
Author(s):  
Edward T PARKIN ◽  
Anthony J TURNER ◽  
Nigel M HOOPER
Keyword(s):  
Light Scattering ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Annexin V ◽  
Density Fraction ◽  
Calcium Dependent ◽  
Membrane Complex ◽  
Isolation And Characterization ◽  
Insoluble Complex ◽  
Marker Proteins

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5´-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca2+-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the metal chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.

Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array

2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Zachary K. Lindstrom ◽  
Steven J. Brewer ◽  
Melanie A. Ferguson ◽  
Sandra H. Burnett ◽  
Brian D. Jensen
Keyword(s):  
Gene Therapy ◽  
Nucleic Acids ◽  
Propidium Iodide ◽  
Hela Cells ◽  
Human Cells ◽  
Advantages And Disadvantages ◽  
Culture Cells ◽  
High Viability ◽  
Promising Area ◽  
Hela Cell Culture

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78–91% viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

METABOLISM OF LEPTOSPIRES: II. THE ACTION OF 8-AZAGUANINE

1967 ◽  
Vol 13 (12) ◽  
pp. 1621-1629 ◽  
Author(s):  
Russell C. Johnson ◽  
Palmer Rogers
Keyword(s):  
Nucleic Acids ◽  
Ribonucleic Acid ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Relative Increase ◽  
Purine Biosynthesis ◽  
Low Concentrations

Both the pathogen Leptospira pomona and the saprophyte L. biflexa Patoc I can convert exogenous adenine, guanine, and 8-azaguanine to the corresponding nucleotide and incorporate them into nucleic acids. L. pomona is inhibited by low concentrations of 8-azaguanine (50 μg/ml) and this inhibition is associated with less than a 5% replacement of the ribonucleic acid (RNA) guanine residues by the analogue. Guanine possessed the highest activity for antagonizing the inhibitory effect of 8-azaguanine. The biosynthetic process of L. pomona most affected by the analogue was a relative increase in RNA synthesis. The analogue-resistant L. biflexa incorporated 1/10 as much 8-azaguanine as L. pomona. The higher rate of purine biosynthesis, in addition to the lesser amount of 8-azaguanine incorporated, may account for the analogue resistance of L. biflexa.

scholarly journals The effects of hinokitiol on human cells revealed by a proteomic approach

2012 ◽  
Vol 32 (3) ◽  
pp. 137-143
Author(s):  
Seido Ooka ◽  
Toshiyuki Sato ◽  
Mitsumi Arito ◽  
Hiromasa Nakano ◽  
Yukiko Takakuwa ◽  
...  
Keyword(s):  
Human Cells ◽  
Proteomic Approach

scholarly journals Observation of nucleic acids inside living human cells by in-cell NMR spectroscopy

2020 ◽  
Vol 17 (0) ◽  
pp. 36-41
Author(s):  
Yudai Yamaoki ◽  
Takashi Nagata ◽  
Tomoki Sakamoto ◽  
Masato Katahira
Keyword(s):  
Nmr Spectroscopy ◽  
Nucleic Acids ◽  
Human Cells

scholarly journals High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events.

1983 ◽  
Vol 97 (3) ◽  
pp. 838-848 ◽  
Author(s):  
J A Kleinschmidt ◽  
U Scheer ◽  
M C Dabauvalle ◽  
M Bustin ◽  
W W Franke
Keyword(s):  
Rana Temporaria ◽  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
High Mobility ◽  
High Mobility Group ◽  
Monomeric Form ◽  
Hmg Proteins ◽  
Amphibian Species ◽  
Large Oocyte ◽  
Calf Thymus

Oocytes of several amphibian species (Xenopus laevis, Rana temporaria, and Pleurodeles waltlii) contained a relatively large pool of nonchromatin-bound, soluble high mobility group (HMG) protein with properties similar to those of calf thymus proteins HMG-1 and HMG-2 (protein HMG-A; A, amphibian). About half of this soluble HMG-A was located in the nuclear sap, the other half was recovered in enucleated ooplasms. This protein was identified by its mobility on one- and two-dimensional gel electrophoresis, by binding of antibodies to calf thymus HMG-1 to polypeptides electrophoretically separated and blotted on nitrocellulose paper, and by tryptic peptide mapping of radioiodinated polypeptides. Most, if not all, of the HMG-A in the soluble nuclear protein fraction, preparatively defined as supernatant obtained after centrifugation at 100,000 g for 1 h, was in free monomeric form, apparently not bound to other proteins. On gel filtration it eluted with a mean peak corresponding to an apparent molecular weight of approximately 25,000; on sucrose gradient centrifugation it appeared with a very low S value (2-3 S), and on isoelectric focusing it appeared in fractions ranging from pH approximately 7 to 9. This soluble HMG-A was retained on DEAE-Sephacel but could be eluted already at moderate salt concentrations (0.2 M KCl). In oocytes of various stages of oogenesis HMG-A was accumulated in the nucleus up to concentrations of approximately 14 ng per nucleus (in Xenopus), corresponding to approximately 0.2 mg/ml, similar to those of the nucleosomal core histones. This nuclear concentration is also demonstrated using immunofluorescence microscopy. When antibodies to bovine HMG-1 were microinjected into nuclei of living oocytes of Pleurodeles the lateral loops of the lampbrush chromosomes gradually retracted and the whole chromosomes condensed. As shown using electron microscopy of spread chromatin from such injected oocyte nuclei, this process of loop retraction was accompanied by the appearance of variously-sized and irregularly-spaced gaps within transcriptional units of chromosomal loops but not of nucleoli, indicating that the transcription of non-nucleolar genes was specifically inhibited by this treatment and hence involved an HMG-1-like protein. These data show that proteins of the HMG-1 and -2 category, which are usually chromatin-bound components, can exist, at least in amphibian oocytes, in a free soluble monomeric form, apparently not bound to other molecules. The possible role of this large oocyte pool of soluble HMG-A in early embryogenesis is discussed as well as the possible existence of soluble HMG proteins in other cells.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Analysis of the intermediate size proteoglycans from the developing chick limb buds11Presented in part at 72nd Annual Meeting of the American Society of Biological Chemists, St Louis, Missouri, U.S.A. 31 May to 4 June, 1981.

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 61-74
Author(s):  
Nagaswamisri Vasan
Keyword(s):  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Limb Bud ◽  
Side Chain ◽  
Intermediate Size ◽  
Proteoglycan Aggregates ◽  
American Society ◽  
Hyaluronic Acid Binding

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

Sign in / Sign up

Export Citation Format

Share Document