SEARCH · Search PubMed

Results for “modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources

PubMed1999

Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.

Ascorbic Acid↗

PubMed2004

Modification of beta-lactoglobulin by oligofructose: impact on protein adsorption at the air-water interface.

Maillard products of beta-lactoglobulin (betaLg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of betaLg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidine-HCl. The modification leads to lowering of the denaturation temperature by 5 degrees C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified betaLg exerts a lower surface pressure than native betaLg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-betaLg. Compared to native betaLg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications.

Adsorption↗

PubMed2003

Mass spectroscopic characterization of protein modification by 4-hydroxy-2-(E)-nonenal and 4-oxo-2-(E)-nonenal.

The modification of proteins by 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) was investigated using mass spectroscopic approaches. Electrospray ionization MS analysis of HNE- and ONE-treated myoglobin and apomyoglobin revealed that the latter more "open" protein structure resulted in more extensive modification. Reductive methylation of Lys residues halved the extent of modification, implicating the importance of adduction of HNE and ONE to both His and Lys residues. HPLC-MS/MS analysis of tryptic and chymotryptic peptides of HNE- or ONE-adducted apomyoglobin was aided by the knowledge of structures previously elucidated through model reactions. In the case of HNE, the adducts detected were the HNE-His Michael adduct (on H24, H36, H64, and H113), its dehydrated form (on H36), and the HNE-Lys pyrrole adduct (on K16, K42, K45, K145, and K147). In the case of the more reactive ONE, the adducts detected were the ONE-His Michael adduct (on H24), the ONE-Lys pyrrolinone adduct (on K16 and K145), and the ONE-His-Lys pyrrole cross-link (linking K16 to H24 in the C(5) peptide). Although previous analyses of tryptic peptides yielded findings about the nature of His modification, the current chymotryptic peptide analysis produced the first structural characterization of Lys modification on intact proteins by HNE and ONE using mass spectrometry.

Aldehydes↗

PubMed2001

Is that what I said?: witnesses' responses to interviewer modifications.

Modifications occur when interviewers contradict statements made by witnesses or imply that witnesses provided information that they (interviewers) did not provide. Because of their suggestive nature, modifications threaten the reliability of investigative interviews. This study investigated developmental differences in witnesses' responses to modifications during interviews as well as in inclusion of modified misinformation in subsequent answers. Preschool, elementary school, and college students were interviewed about a video presentation. In the experimental conditions, the interviewer contradicted information about the video provided by the participants. Participants then answered two sets of follow-up questions: one immediately following the interview and another 6-8 days later. Results indicated that participants were more likely to ignore modifications than to correct or agree with them. Adult participants were most likely to disagree with modifications. Preschoolers were most likely to incorporate modified misinformation into subsequent answers. Implications of these findings for investigative interviews are discussed.

Adolescent↗

PubMed2003

Chemical modification of catalytically essential functional groups of NAD-dependent hydrogenase from Ralstonia eutropha H16.

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions led to the modification of additional His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the activity of the hydrogenase. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.

Cupriavidus necator↗

PubMed1993

Separate and combined effects of methylphenidate and behavior modification on boys with attention deficit-hyperactivity disorder in the classroom.

This study evaluated the separate and combined effects of behavior modification and 2 doses of methylphenidate (MPH; 0.3 and 0.6 mg/kg) compared with baseline (no behavior modification and a placebo) on the classroom behavior and academic performance of 31 ADHD (attention deficit-hyperactivity disorder) boys attending a summer treatment program. Results revealed significant effects of both interventions, with the mean effect size of medication being more than twice as great as that of behavior modification. Relatively small incremental value was gained by the higher dose of medication or the addition of behavior modification, compared with the effects of the low dose of MPH. In contrast, the addition of either dose of MPH resulted in improvement beyond the effects of behavior modification alone. These group effects reflected those obtained in analyses of individual differences. Furthermore, comparisons of individual responsiveness showed that boys who responded to one treatment also responded to the other.

Attention Deficit Disorder with Hyperactivity↗

PubMed2002

Spike-timing-dependent synaptic modification induced by natural spike trains.

The strength of the connection between two neurons can be modified by activity, in a way that depends on the timing of neuronal firing on either side of the synapse. This spike-timing-dependent plasticity (STDP) has been studied by systematically varying the intervals between pre- and postsynaptic spikes. Here we studied how STDP operates in the context of more natural spike trains. We found that in visual cortical slices the contribution of each pre-/postsynaptic spike pair to synaptic modification depends not only on the interval between the pair, but also on the timing of preceding spikes. The efficacy of each spike in synaptic modification was suppressed by the preceding spike in the same neuron, occurring within several tens of milliseconds. The direction and magnitude of synaptic modifications induced by spike patterns recorded in vivo in response to natural visual stimuli were well predicted by incorporating the suppressive inter-spike interaction within each neuron. Thus, activity-induced synaptic modification depends not only on the relative spike timing between the neurons, but also on the spiking pattern within each neuron. For natural spike trains, the timing of the first spike in each burst is dominant in synaptic modification.

Action Potentials↗

PubMed2002

Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component.

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

Fluorescent Antibody Technique↗

PubMed2026

DREAMS illuminates spatial DNA and RNA modification landscapes.

DNA and RNA modifications regulate gene expression and RNA processing, but their spatial organization in complex tissues remains elusive. Here we developed DNA RNA Elements Areal Mass Spectrometry (DREAMS), a mass spectrometry imaging platform that spatially maps diverse nucleic acid modifications simultaneously. Applying DREAMS to TET-deficient mouse brains (Tet1Δ/Δ and triple Tet1/2/3Δ/Δ), we uncover TET1's unexpected role in modulating N1-methyladenosine (m1A), a pivotal RNA modification. While DREAMS reveals broad modification landscapes altered across TET knockouts, we identify TET1-mediated changes in m1A that correlate with transcriptome alterations. Our work establishes DREAMS as a transformative tool for spatial epigenomics/epitranscriptomics and suggests that TET enzymes could influence multiple DNA and RNA modifications with potential gatekeeping roles in nucleic acid regulation.

Animals↗

PubMed2025

Uncovering hidden protein modifications with native top-down mass spectrometry.

Protein modifications drive dynamic cellular processes by modulating biomolecular interactions, yet capturing these modifications within their native structural context remains a significant challenge. Native top-down mass spectrometry promises to preserve the critical link between modifications and interactions. However, current methods often fail to detect uncharacterized or low-abundance modifications, limiting insights into proteoform diversity. To address this gap, we introduce precise and accurate Identification Of Native proteoforms (precisION), an interactive end-to-end software package that leverages a robust, data-driven fragment-level open search to detect, localize and quantify 'hidden' modifications within intact protein complexes. Applying precisION to four therapeutically relevant targets-PDE6, ACE2, osteopontin (SPP1) and a GABA transporter (GAT1)-we discover undocumented phosphorylation, glycosylation and lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map of GAT1. As an open-source software package, precisION offers an intuitive means for interpreting complex protein fragmentation data. This tool will empower the community to unlock the potential of native top-down mass spectrometry, advancing integrative structural biology, molecular pathology and drug development.

Mass Spectrometry↗

PubMed2004

The structure of the TrmE GTP-binding protein and its implications for tRNA modification.

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.

Amino Acid Sequence↗

PubMed2001

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.

Animals↗

PubMed1997

Derivatives of activated H-ras lacking C-terminal lipid modifications retain transforming ability if targeted to the correct subcellular location.

To examine the ability of ras to activate signal transduction pathways in the absence of lipid modifications, fusion proteins were constructed that target rasWT or activated ras61L to cellular membranes as integral membrane proteins, using the first transmembrane domain of the E1 protein of avian infectious bronchitis virus (IBV), which contains a cis-Golgi targeting signal. Golgi-targeted derivatives of activated ras were completely inactive in transformation assays. However, when examined in focus formation assays, transformation of NIH3T3 cells were seen with derivatives of ras61L containing a mutated E1 targeting sequence that results in plasma membrane localization. Removal of the lipid modification sites in and upstream of the CAAX motif did not abrogate the transforming activity of plasma membrane-localized ras61L derivatives, indicating that these lipid modifications are not essential for ras activity, as long as the protein is correctly localized to the plasma membrane. Interestingly, the activity of integral membrane versions of ras61L was strictly dependent on a minimum distance between the transmembrane domain anchor region and the coding sequence of ras. Derivatives with only a 3-amino acid linker were inactive, while linkers of either 11- or 22-amino acids were sufficient to restore transforming activity. These results demonstrate that: (1) activated ras targeted to Golgi membranes is unable to cause transformation; (2) lipid modifications at the C-terminus are not required for the transforming activity of plasma membrane-anchored ras61L derivatives, and serve primarily a targeting function; (3) a transmembrane domain can effectively substitute for C-terminal modifications that would normally target ras to the inner surface of the plasma membrane, indicating that ras61L does not need to reversibly dissociate from the membrane as might be allowed by the normal lipidation; and (4) in order to function properly, there exists a critical distance that the ras protein must reside from the plasma membrane.

3T3 Cells↗

PubMed2001

Regulation of G proteins by covalent modification.

Heterotrimeric G protein alpha,beta, and gamma subunits are subject to several kinds of co- and post-translational covalent modifications. Among those relevant to G protein-coupled receptor signaling in normal cell function are lipid modifications and phosphorylation. N-myristoylation is a co-translational modification occurring for members of the G(i) family of Galpha subunits, while palmitoylation is a post-translational modification that occurs for these and most other Galpha subunits. One or both modifications are required for plasma membrane targeting and contribute to regulating strength of interaction with the Gbetagamma heterodimer, effectors, and regulators of G protein signaling (RGS proteins). Galpha subunits, including those with transforming activity, are often inactive when unable to be modified with lipids. The reversible nature of palmitoylation is intriguing in this regard, as it lends itself to a regulation integrated with the activation state of the G protein. Several Galpha subunits are substrates for phosphorylation by protein kinase C and at least one is a substrate for phosphorylation by the p21-activated protein kinase. Phosphorylation in both instances inhibits the interactions of these subunits with the Gbetagamma heterodimer and RGS proteins. Several Galpha subunits are also substrates for tyrosine phosphorylation. A Ggamma subunit is phosphorylated by protein kinase C, with the consequence that it interacts more tightly with a Galpha subunit but less well with an effector.

Acylation↗

PubMed2001

Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines.

The reactive cysteines in H-ras are subject to oxidative modifications that potentially alter the cellular function of this protein. In this study, purified H-ras was modified by thiol oxidants such as hydrogen peroxide (H(2)O(2)), S-nitrosoglutathione, diamide, glutathione disulphide (GSSG) and cystamine, producing as many as four charge-isomeric forms of the protein. These results suggest that all four reactive cysteines of H-ras are potential sites of regulatory modification reactions. S-nitrosylated and S-glutathiolated forms of H-ras were identified by protocols that depend on separation of alkylated proteins on electrofocusing gels. S-nitrosoglutathione could S-nitrosylate H-ras on four cysteine residues, while reduced glutathione (GSH) and H(2)O(2) mediate S-glutathiolation on at least one cysteine of H-ras. Either GSSG or diamide S-glutathiolated at least two cysteine residues of purified H-ras. Iodoacetic acid reacts with three cysteine residues. In intact NIH-3T3 cells, wild-type H-ras was S-glutathiolated by diamide. Similarly, cells expressing a C118S mutant or a C181S/C184S double mutant of H-ras were S-glutathiolated by diamide. These results suggest that H-ras can be S-glutathiolated on multiple thiols in vivo and that at least one of these thiols is normally lipid-modified. In cells treated with S-nitrosocysteine, evidence for both S-nitrosylated and S-glutathiolated H-ras was obtained and S-nitrosylation was the predominant modification. These results show that oxidative modification of H-ras can be extensive in vivo, that both S-nitrosylated and S-glutathiolated forms may be important, and that oxidation may occur on reactive cysteines that are normally targeted for lipid-modification reactions.

3T3 Cells↗

PubMed2001

Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Aldehyde Reductase↗

PubMed1977

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue.

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75-1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [(14)C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the (14)C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

Binding, Competitive↗

PubMed1991

Dissociation of lethal toxicity and enzymic activity of notexin from Notechis scutatus scutatus (Australian-tiger-snake) venom by modification of tyrosine residues.

Notexin from Notechis scutatus scutatus snake venom was subjected to tyrosine modification with p-nitrobenzenesulphonyl fluoride (NBSF), and four modified derivatives were separated by h.p.l.c. The results of amino acid analysis and sequence determination revealed that only Tyr-7, Tyr-70 and Tyr-77 were modified in notexin. Modification of Tyr-7 resulted in decreases in lethal toxicity and enzymic activity by 70.2% and 22.7% respectively. Conversely, modification of Tyr-77 caused a 1.8-fold increase in enzymic activity, in contrast with the loss of 52.5% of lethality. A drastic decrease in lethal toxicity was observed when both Tyr-7 and Tyr-70 were modified, whereas the enzymic activity decreased by only 35.8%. Likewise, the derivative in which Tyr-7 and Tyr-77 were modified retained 44.4% of enzymic activity, but showed a marked decrease in lethal toxicity. It is obvious that modification of tyrosine residues causes a decrease in lethal toxicity of notexin, which does not directly correlate with the change in enzymic activity. On the other hand, the antigenicity of NBS derivatives remained unchanged. The modified derivatives retained their affinity for Ca2+, indicating that the modified tyrosine residues did not participate in Ca2+ binding. These results indicate that modification of tyrosine residues can differentially influence the enzymic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one being responsible for the catalytic activity and the other associated with its lethal effect.

Amino Acid Sequence↗