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PubMed1994

Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Adenosine Diphosphate Ribose↗
PubMed1985

Protein modification and its biological role.

The modifications present on a polypeptide play an important role in determining its eventual fate. Modifications, particularly proteolysis, are important in the generation of biological activity. Modifications are used to "target" particular polypeptides to specific cellular locations. Protein modification also plays a role in determining the rate of polypeptide degradation. Cells have developed elaborate systems for the modification of their proteins because these modifications serve important biological functions.

Amino Acid Sequence↗
PubMed1990

Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes.

We have investigated whether unmodified yeast phenylalanine transfer RNA as well as one of its precursors containing an intron of nineteen nucleotides in the anticodon (pre-tRNA-Phe) can become substrates for selected tRNA modification enzymes present in a eukaryotic cell. This study was done by microinjecting into the cytoplasm of Xenopus laevis oocytes transcripts completely deprived of the naturally occurring modified nucleotides; these were obtained in vitro from appropriate synthetic genes under the control of bacteriophage T7 promoter. During the in vitro transcription, 32P labels were introduced with the guanosine triphosphate thus allowing easy detection of guanosine modifications in tRNA by two-dimensional chromatography after complete digestion into 5'-mononucleotides by nuclease P1. Results indicate that modifications occur on five guanosines (at positions 10, 26, 34, 37 and 46) in yeast tRNA-Phe and only on three guanosines (at 10, 26 and 46) in yeast precursor tRNA-Phe. These are the modifications expected from the known nucleotide sequences of naturally occurring Xenopus and yeast tRNA-Phe, i.e. N2-methyl-G10, N2,N2-dimethyl-G26, 2'-O-methyl-G34, N1-methyl-G37 or Y nucleoside-37 and N7-methyl-G46. The rates of modifications occurring in the two kinds of tRNA-Phe are faster in the intron-less tRNA-Phe than in the intron-containing tRNA-Phe. However quantitative modifications are only observed after as long as 75 h incubation in the oocytes.

Animals↗
PubMed1994

Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group.

The role of carboxyl group in the catalytic action of xylanase (M(r) 35,000) from an alkalothermophilic Bacillus sp. was delineated through kinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M-1 min-1. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar Km and lower kcat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Deshpande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172-177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.

Amino Acids↗
PubMed1983

Kinetic analysis of biphasic protein modification reactions. Cooperative effects.

A mathematical treatment of protein modification reactions is presented, and it is shown that in these cases protein modification is described by a summation of exponential functions of reaction time, the number of exponentials being equal to the number of modified protein species. It is shown that, in cases of protein modification cooperativity, there is a strict dependence of the coefficients of the multiexponential modification equation on the constants of the same equation. The conditions necessary for a reduction of a multiexponential protein modification equation to one of a summation of two exponentials only are examined. The possible formulae for the coefficients of a two-exponential-summation equation, used to describe the modification of protein models with two, three or four modifiable residues (as well as some aspects of models with five and six modifiable residues) per protein molecule are derived. It is seen that the number of such coefficients is severely limited. The most frequently obtained formula for the lower stoichiometric coefficient of a two-exponential-summation equation Is Aka/(ka-kb), where ka and kb are the constants of the two exponentials of the equation, and A is a constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymes↗
PubMed2005

Characterization of advanced glycation end products: mass changes in correlation to side chain modifications.

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains are supposed to be involved in the pathogenesis of several diseases; therefore, the effects of AGEs on cells are the objective of numerous investigations. Because AGE modifications are an extremely heterogeneous group of side chain modifications, the exact characterization of an AGE-modified protein is impossible. To gain a deeper understanding about AGE formation kinetics and structures, AGEs can be characterized with respect to the degree of modification, specific side chain modifications, absorbance and fluorescence characteristics, and changes in the protein structure and molecular weight. For this study, human serum albumin (HSA)-AGEs derived from different concentrations of glucose, methyl glyoxal, and glyoxylic acid were used. The molecular mass of the obtained AGEs was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass data were compared with earlier results concerning the degree of lysine and arginine side chain modifications and AGE-specific fluorescence and absorbance data. The molecular masses were found to gradually increase with increasing concentrations of the individual modifier without reaching a plateau. The mass increase correlates very well with the AGE-specific absorbance at 360 nm and with the degree of side chain modifications. The mass spectrometric data prove, for the first time, that an increasing absorbance at 360 nm is directly correlated to a mass increase during the AGE formation process.

Glycation End Products, Advanced↗
PubMed2006

Effects of lifestyle modification on the progression of coronary atherosclerosis, autonomic function, and angina--the role of GNB3 C825T polymorphism.

BACKGROUND: Given the multimodal medical and interventional treatment options in coronary artery disease, the additional value of intensified lifestyle modification is unclear. We have therefore examined the effects of lifestyle modification on top of current treatment and also associated with the GNB3 C825T polymorphism, which has established association to sympathetic activation and the precipitation of angina. METHODS: One hundred one patients with established coronary artery disease were randomized to a 1-year lifestyle modification group (lifestyle group [LG]) or an advice group. Risk factors, coronary calcification (electron beam tomography), heart rate variability, baroreflex sensitivity, anginal symptoms, and quality of life (QOL) were assessed on entry and after 1 year. RESULTS: Patients in LG had excellent program adherence, but lifestyle modification had no impact on metabolic risk factors and coronary calcification. Changes in heart rate, heart rate variability, and blood pressure were only slightly favoring LG. Baroreflex sensitivity increased by 2 (0.79-3.13) ms/mm Hg in the LG but decreased by -0.10 (-1.11 to 0.92) in the advice group (P = .013). Lifestyle modification led to improved physical QOL, reductions of anginal attacks (-54% vs 11%, P = .01), and dose reductions in 30% of anti-ischemic medications (P = .004). *825T allele carriers had a more pronounced reduction of heart rate and improvement of angina and QOL. The beneficial effect on reduction of medication was seen in *825T allele carriers only. CONCLUSIONS: In the presence of modern treatments, comprehensive lifestyle modification provides no additional benefits on progression of atherosclerosis but improves autonomic function, angina, and QOL with concomitant reduced need of medication. These responses are more pronounced in GNB3*825T allele carriers.

Aged↗
PubMed2006

The analysis of histone modifications.

The biological function of many proteins is often regulated through posttranslational modifications (PTMs). Frequently different modifications influence each other and lead to an intricate network of interdependent modification patterns that affect protein-protein interactions, enzymatic activities and sub-cellular localizations. One of the best-studied class of proteins that is affected by PTMs and combinations thereof are the histone molecules. Histones are very abundant, small basic proteins that package DNA in the eukaryotic nucleus to form chromatin. The four core-histones are densely modified within their first 20-40 N-terminal amino acids, which are highly evolutionary conserved despite playing no structural role. The modifications are thought to constitute a histone code that is used by the cell to encrypt various chromatin conformations and gene expression states. The analysis of modified histones can be used as a model to dissect complex modification patterns and to investigate their molecular functions. Here we review techniques that have been used to decipher complex histone modification patterns and discuss the implication of these findings for chromatin structure and function.

Amino Acid Sequence↗
PubMed2006

Chemical modification of siRNAs to improve serum stability without loss of efficacy.

Development of RNA interference as a novel class of therapeutics requires improved pharmacokinetic properties of short interfering RNA (siRNA). To confer enhanced serum stability to Sur10058, a hyperfunctional siRNA which targets survivin mRNA, a systematic modification at the 2'-sugar position and phosphodiester linkage was introduced into Sur10058. End modification of three terminal nucleotides by 2'-OMe and phosphorothioate substitutions resulted in a modest increase in serum stability, with 3' end modification being more effective. Alternating modification by 2'-OMe substitution significantly stabilized Sur10058, whereas phosphorothioate modification was only marginally effective. Through various combinations of 2'-OMe, 2'-F and phosphorothioate modifications that were directed mainly at pyrimidine nucleotides, we have identified several remarkably stable as well as efficient forms of Sur10058. Thus, our results provide an effective means to stabilize siRNA in human serum without compromising the knockdown efficiency. This advancement will prove useful for augmenting the in vivo potency of RNA interference.

Base Sequence↗
PubMed2005

Fluoride modification effects on osteoblast behavior and bone formation at TiO2 grit-blasted c.p. titanium endosseous implants.

Increasing bone formation at endosseous titanium implants may be achieved by modification of topographically enhanced surfaces. The aim of this study was to determine the effect of fluoride ion modification of TiO2 grit-blasted, c.p. titanium implants on osteoblastic differentiation and interfacial bone formation by parallel in vitro and in vivo investigations. Human mesenchymal stem cells (Osiris Therapeutics, Inc.) were cultured on TiO2 grit-blasted c.p.titanium disks with and without fluoride ion modification. Cell adhesion, proliferation, and osteoblastic gene expression was measured by scanning electron microscopy, tritiated-thymidine uptake into insoluble DNA, and reverse transcription polymerase chain reaction detection of mRNAs encoding collagen 1, osteopontin, bone sialoprotein, osteocalcin and BMP-2. After 24 h, there were no differences in cell adhesion among the surfaces tested. Fluoride-treated surfaces supported greater proliferation and increased bone sialoprotein and BMP-2 expression. Additionally, 12 TiO2 grit-blasted and 12 fluoride ion modified implants were placed randomly into medial and distal osteotomies prepared in the tibia of 300 g Sprague Dawley rats. After 21 days, the tibiae were harvested and 100 microm ground sections were examined by backscatter scanning electron microscopy. The bone-to-implant contact formed at TiO2 grit-blasted and fluoride-treated versus TiO2 grit-blasted surfaces was 55.45% versus 34.21% (p<0.027), respectively. Fluoride ion modification of the TiO2 grit-blasted surface enhanced osteoblastic differentiation in vitro and interfacial bone formation in vivo. This parallel in vitro and in vivo investigation demonstrates that fluoride ion modification enhanced osteoblastic differentiation and interfacial bone formation. The mechanism(s) by which fluoride ion modification of c.p.titanium enhanced osteoblastic differentiation and osseointegration merit careful investigation.

Animals↗
PubMed2006

Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.

Amino Acid Sequence↗
PubMed2004

Differential expression of histone post-translational modifications in acute myeloid and chronic lymphocytic leukemia determined by high-pressure liquid chromatography and mass spectrometry.

The post-translational modification of the core histones is critical to the regulation of chromatin structure. Traditional methods for the determination of histone modification utilize immunoassay techniques to determine the extent and site of post-translational modification. These methods, though sensitive, require site-specific antibodies. This manuscript describes the application of reverse-phase high-pressure liquid chromatography and mass spectrometry (LC-MS) to analyze global modification levels of core histones. The method is fast, sensitive, and easily automated. Furthermore, the technique gives the global patterns of modification for all four core histones in a single experiment. The LC-MS method was optimized using histones extracted from bovine thymus. These methods were then applied to the characterization of changes in histone modification in acute myeloid leukemia (AML) cell lines treated with histone deacetylase (HDAC) inhibitors. Dose-dependent changes in the distribution of modified core histones were observed. These results were validated in primary leukemia cells from patients with refractory or relapsed AML or chronic lymphocytic leukemia (CLL) treated on a Phase I clinical trial of the HDAC inhibitor depsipeptide. An increase in the relative abundance of specific acetylated forms of histone H4 was readily observable in these patients at intervals of 4 and 24 h after treatment.

Animals↗
PubMed2005

Cranial vault modification as a cultural artifact: a comparison of the Eurasian steppes and the Andes.

This paper details the practice of intentional cranial vault modification in the Eurasian steppes as well as in the pre-Columbian Andes focusing on the similarities and differences in how the practice was used to respond to changes in society. The appearance of vault modification in the steppes and the forms seen in the cemeteries of the Syr Darya and Amu Darya River deltas are discussed. Temporal changes in the pattern of modification are also investigated, especially the dramatic homogenization of the custom resulting from the conquests of the Huns. This is contrasted with incidences of cranial modification in the south-central Andes, including the appearance of deliberate head shaping as well as shifts in the practice during the expansion of the Bolivian Altiplano state of Tiwanaku. Similarities in the use of cranial vault modification between these unrelated areas and in the alterations of the practice resulting from foreign contact are considered in light of vault modification's role as a malleable cultural artifact.

Adult↗
PubMed2005

In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants.

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.

Aldehydes↗
PubMed2006

tRNA's wobble decoding of the genome: 40 years of modification.

The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.

Anticodon↗
PubMed2006

Receptive-field modification in rat visual cortex induced by paired visual stimulation and single-cell spiking.

Experience-dependent plasticity of visual cortical receptive fields (RFs) involves synaptic modifications in the underlying neural circuits, but the site and mechanism of these modifications remain to be elucidated. Using in vivo whole-cell recordings, we show that pairing visual stimulation at a given retinal location with spiking of a single neuron in developing rat visual cortex induces rapid RF modifications. The time course of the response to the visual stimulus at the paired RF location is altered, with an enhancement of the response preceding the spike time and a reduction following the spike. Such bidirectional modification is consistent with spike timing-dependent plasticity. Response modification also occurs at nearby locations, the direction and magnitude of which are correlated with the change at the paired location. In addition, changes at unpaired locations show a negative correlation with the initial strength of the response, which may facilitate rapid modification of the spatial RF profile.

Animals↗
PubMed2006

Effect of UTP sugar and base modifications on vaccinia virus early gene transcription.

Prior efforts demonstrated that RNA oligonucleotides containing the transcription termination signal UUUUUNU stimulate premature termination of vaccinia virus early gene transcription, in vitro. This observation suggests that viral transcription termination may be an attractive target for the development of anti-poxvirus agents. Since short RNA molecules are readily susceptible to nuclease digestion, their use would require stabilizing modifications. In order to evaluate the effect of both ribose and uracil modifications of the U5NU signal on early gene transcription termination, UTP derivatives harboring modifications to the uracil base, the 2' position of the ribose sugar and the phosphodiester bond were examined in an in vitro vaccinia virus early gene transcription termination system. Incorporation of 4-S-U, 5-methyl-U, 2-S-U, pseudo U and 2'-F-dU into the nascent transcript inhibited transcription termination. 6-aza-U, 2'-amino-U, 2'-azido-U and 2'-O methyl-U inhibited transcription elongation resulting in the accumulation of short transcripts. The majority of the short transcripts remained in the ternary complex and could be chased into full-length transcripts. Initially, derivatives of all uridines in the termination signal were tested. Partial modification of the termination signal reduced termination activity, as well. Introduction of 2'-O methyl ribose to the first three uridines of the U9 termination signal reduced the ability of U9 containing oligonucleotides to stimulate in vitro transcription termination, in trans. Further modifications eliminated this activity. Thus, viral early gene transcription termination demonstrates a rigorous requirement for a U5NU signal that is unable to tolerate modification to the base or sugar. Additionally, VTF was shown to enhance transcription elongation through the T9 sequence in the template. These results suggest that VTF may play a subtle role in early gene transcription elongation in addition to its known function in mRNA cap formation, early gene transcription termination and intermediate gene transcription initiation.

Animals↗
PubMed2000

Labeling bias and attitudes toward behavior modification revisited.

Twenty-three years ago, Woolfolk, Woolfolk and Wilson (1977, Journal of Consulting and Clinical Psychology, 45, 184-191) described a study about labeling bias and attitudes toward behavior modification. When videotaped depictions of behavior modification procedures were described as "humanistic education", college students rated them more favorably than when the same procedures were called "behavior modification". One implication of this study is that behavioral terminology may be perceived as dehumanizing by potential consumers, leading to lower acceptance of the approach regardless of its effectiveness. With lower acceptance comes the risk of under utilization. More than two decades have passed since the Woolfolk et al. study was published so we felt the time was right to repeat the experiment with a new generation of students. In our study, two groups of college undergraduates (N = 144) gave their opinions about a 10 minute videotape that showed a therapist working with an autistic child. The therapist used behavior modification procedures to treat the child, except in one case the procedures were described as "humanistic educative therapy", and in the other as "behavior modification". An appropriate rationale was provided for each of the two conditions. Unlike Woolfolk et al., we found no differences in how the two depictions were evaluated. In both cases, they were perceived favorably regardless of what they were called, as were the personal qualities of the therapist. These results suggest that negative attitudes toward behavior modification have weakened over the last two decades, and there is wider pubic acceptance of behavioral treatments now than there was then. Implications of the study are briefly discussed.

Adult↗