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PubMed2006

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Catalytic Domain↗
PubMedDate not supplied

Establishment of a hybrid SalI-HgiDII type II restriction-modification system.

In the SalI system, endonuclease activity can be only achieved in the presence of a functional modification gene. Thus, the DNA methyltransferase is involved in the control of restriction. By fusion of the restriction gene of the SalI system to the modification gene of the isospecific HgiDII system a hybrid type II restriction-modification system was created. Although in the hybrid situation the level of endonuclease activity was significantly lower than in the natural system, the HgiDII modification enzyme clearly supports SalI restriction. The mechanism by which the two isospecific methyltransferases control restriction is currently under study.

DNA Modification Methylases↗
PubMed1979

Behavior modification in the treatment of obesity. The problem of maintaining weight loss.

The introduction of behavior modification in the treatment of obesity a decade ago resulted in a substantial increase in weight loss of persons treated for mild to moderate obesity. It has been hoped that this increased effectiveness of treatment would extend also to maintenance of weight loss, and the first controlled clinical trial suggested that it did. This article reports the results of (1) a five-year follow-up of this first trial and (2) all follow-up studies of behavior modification for obesity yet conducted, six published and four previously unpublished. A new method of data analysis shows that weight losses are only modestly maintained, although the question of how their maintenance compares with that of other treatments cannot be answered because comparable data on other treatments are not available. In contrast to the vast amount of work on the initiation and generalization of behavior change, study of its maintenance has been a neglected area of behavior modification. We suggest that this area constitutes a new frontier for research in behavior modification.

Behavior Therapy↗
PubMed1995

Changes in myocardial perfusion abnormalities by positron emission tomography after long-term, intense risk factor modification.

OBJECTIVE: To quantify changes in size and severity of myocardial perfusion abnormalities by positron emission tomography (PET) in patients with coronary artery disease after 5 years of risk factor modification. DESIGN: Randomized controlled trial. SETTING: Outpatient community setting. INTERVENTION: Randomization of patients to risk factor modification consisting of very low-fat vegetarian diet, mild to moderate exercise, stress management, and group support (experimental group, n = 20) or to usual care by their own physicians, consisting principally of antianginal therapy (control group, n = 15). MAIN OUTCOME MEASURES: Quantitative coronary arteriography and PET at baseline and 5 years after randomization. Automated, objective measures of size and severity of perfusion abnormalities on rest-dipyridamole PET images and of stenosis severity on arteriograms were made by computer algorithms. RESULTS: Size and severity of perfusion abnormalities on dipyridamole PET images decreased (improved) after risk factor modification in the experimental group compared with an increase (worsening) of size and severity in controls. The percentage of left ventricle perfusion abnormalities outside 2.5 SDs of those of normal persons (based on 20 disease-free individuals) on the dipyridamole PET image of normalized counts worsened in controls (mean +/- SE, + 10.3% +/- 5.6%) and improved in the experimental group (mean +/- SE, -5.1% +/- 4.8%) (P = .02); the percentage of left ventricle with activity less than 60% of the maximum activity on the dipyridamole PET image of normalized counts worsened in controls (+13.5% +/- 3.8%) and improved in the experimental group (-4.2% +/- 3.8%) (P = .002); and the myocardial quadrant on the PET image with the lowest average activity expressed as a percentage of maximum activity worsened in controls (-8.8% +/- 2.3%) and improved in the experimental group (+4.9% +/- 3.3%) (P = .001). The size and severity of perfusion abnormalities on resting PET images were also significantly improved in the experimental group as compared with controls. The relative magnitude of changes in size and severity of PET perfusion abnormalities was comparable to or greater than the magnitude of changes in percent diameter stenosis, absolute stenosis lumen area, or stenosis flow reserve documented by quantitative coronary arteriography. CONCLUSIONS: Modest regression of coronary artery stenoses after risk factor modification is associated with decreased size and severity of perfusion abnormalities on rest-dipyridamole PET images. Progression or regression of coronary artery disease can be followed noninvasively by dipyridamole PET reflecting the integrated flow capacity of the entire coronary arterial circulation.

Adult↗
PubMed2000

Protein modification during antiviral heat bioprocessing.

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg-white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet- and dry-heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI-MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet-heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys(97), by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality.

Carbohydrates↗
PubMed2000

Influence of surface modifications to titanium on oral bacterial adhesion in vitro.

The influence of surface modifications to titanium on the initial adherence of Porphyromonas gingivalis ATCC33277 and Actinobacillus actinomycetemcomitans ATCC43718 was evaluated. Surface modifications were performed with dry processes including ion implantation (Ca(+), N(+), F(+)), oxidation (anode oxidation, titania spraying), ion plating (TiN, alumina), and ion beam mixing (Ag, Sn, Zn, Pt) with Ar(+) on polished pure titanium plates. Comparatively large amounts of P. gingivalis and A. actinomycetemcomitans adhered to polished titanium plates. The degree of P. gingivalis adhesion showed a positive correlation with surface energy and the amount of calcium-ion adsorption. Adherence of both P. gingivalis and A. actinomycetemcomitans increased on calcium-implanted surfaces compared with polished titanium surfaces, whereas adherence of P. gingivalis was remarkably decreased on alumina-coated surfaces. These findings indicate that titanium implants exposed to the oral cavity require surface modification to inhibit the adherence of oral bacteria, and that surface modification with a dry process is useful in controlling the adhesion of oral bacteria as well as ensuring resistance against wear.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Application of chemical selective cleavage methods to analyze post-translational modification in proteins.

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.

Animals↗
PubMed2003

Cooperative interactions between epigenetic modifications and their function in the regulation of chromosome architecture.

Epigenetic information is encoded by DNA methylation and by covalent modifications of histone tails. While defined epigenetic modification patterns have been frequently correlated with particular states of gene activity, very little is known about the integration level of epigenetic signals. Recent experiments have resulted in the characterization of several epigenetic adaptors that mediate interactions between distinct modifications. These adaptors include methyl-DNA binding proteins, chromatin remodelling enzymes and siRNAs. Complex interactions between epigenetic modifiers and adaptors provide the foundation for the stability of epigenetic inheritance. In addition, they also provide an explanation for the long-range effects of epigenetic mechanisms. We propose that a major aspect of epigenetic regulation lies in the modification of chromosome architecture and that local changes in gene expression would be secondary consequences. This view is consistent with many results from recent genomic analyses.

Animals↗
PubMed2005

The partial retro-inverso modification: a road traveled together.

In the mid-1970s, Dr. Murray Goodman was interested in a reversed peptide bond as a surrogate to understand the functional role of the amide bond in aspartame, a dipeptide sweetener. Very soon, realizing the breath and potential of this modification, Murray expanded this activity into a full program and I was fortunate to be part of it. Together we formulated new concepts such as the partially modified retro-inverso and end-group modified retro-inverso transformations, tested hypotheses, generated novel nomenclature, developed synthetic routes, characterized the preferred conformations of the unique building blocks employed in this modification, the gem-diaminoalkyl and the C2-substituted malonyl residues, and studied the biological activity of retro-inverso isomers of bioactive peptides. In the early 1980s several laboratories initiated extensive research targeted at the retro-inverso modification. The revival of this field led to new applications, new methods of synthesis, and new insights on the conformational and topological properties of the retro-inverso modification. Among the fields that embraced the retro-inverso concept were immunology as pertains to subjects such as synthetic vaccines, immunomodulators, and diagnostic tools, and drug delivery field as pertains to targeted and nontargeted cell permeation vectors loaded with bioactive cargo. Doctor Murray Goodman's sudden death leaves behind not only family, friends, and colleagues, but also an impressive record of scientific achievements among which is the revival of the modern era of the retro-inverso transformation. Murray's numerous contributions, excellent leadership, enthusiastic promotion, and outstanding teachings in this field will carry and illuminate his memory far into the future.

Amino Acid Sequence↗
PubMed2005

Protein structural changes in keratin fibers induced by chemical modification using 2-iminothiolane hydrochloride: a Raman spectroscopic investigation.

For the purpose of investigating in detail the influence of chemical modification using 2-iminothiolane hydrochloride (2-IT) on keratin fibers, the structure of cross-sections at various depths of white human hair, treated with 2-IT and then oxidized, was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. In particular, the beta-sheet and/or random coil content (beta/R) and the alpha-helix (alpha) content in human hair fibers were estimated by amide I band analysis. The S-S band intensity, amide III (unordered) band intensity, and beta/R content existing from the cuticle region to the center of cortex region of virgin white human hair remarkably increased by performing the chemical modification using 2-IT. On the other hand, not only the S-S band intensity, but also S-O band intensity existing throughout the cortex region of the bleached (damaged) white human hair increased by performing chemical modification using 2-IT. In particular, beta/R content existing throughout the cortex region of the bleached white human hair decreased, while the skeletal C-C stretch (alpha) band intensity at 935 cm(-1) and the alpha content remarkably increased. This indicates a secondary structural change from the random coil form to the alpha-helix form in the proteins existing throughout the cortex region. From these experiments, we concluded that the formation of new disulfide (-SS-) groups resulting from chemical modification using 2-IT induced the secondary structural changes of proteins existing throughout the cortex region.

Cross-Linking Reagents↗
PubMed1989

Site-specific laser modification (cleavage) of oligodeoxynucleotides.

Sequence-specific photomodification of oligodeoxynucleotide pAGAGTATTGACTTA ("a target") has been carried out with the aid of complementary fluorescent probes. Such a probe consisted of oligodeoxynucleotide pAATACTCT and a chromophore group attached to its 5' end. Three different derivatives of ethidium bromide were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2). The irradiation induces the following photodamages: target cleavage at the specific binding site with a cutting off of the 8-mer from its 5' end (yield up to 12%), formation of specific covalent adduct target-probe with a yield of 20-70%, and piperidine-sensitive target modifications with a 7-27% yield (for different chromophores). The total yield of specific photodamages of all kinds is 50-80%. The target cleavage and generation of piperidine-sensitive modifications are optically nonlinear processes. Piperidine treatment of the irradiated samples led to specific cleavage of the target with the yield up to 40%. All kinds of observed modifications are not influenced by high concentrations of free radical scavengers: 1.3M tBuOH and 10 mM cystamine. The pattern of cleavage indicates that the most probable position of the chromophore is between T8 and G9 of the target, i.e., the chromophore stacks on top of the last A.T base pair of the duplex. The aggregate of evidence is in agreement with the mechanism of nonlinear photomodification (the cleavage and generation of piperidine-sensitive modifications) based on the transfer of two-photon excitation energy from the chromophore to the target.

Base Sequence↗
PubMed2006

Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk.

A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.

Animals↗
PubMed1993

Investigation of the interaction between enzyme and inhibitor by the combination of chemical modification, electrospray ionization mass spectrometry and frit-fast atom bombardment liquid chromatography/mass spectrometry.

The interaction between enzyme and its inhibitor, hen egg-white lysozyme and tri-N-acetylglucosamine (NAG3), was studied by the combination of chemical modification, enzymatic digestion, electrospray ionization mass spectrometry and frit-fast atom bombardment liquid chromatography/mass spectrometry. Chemical modification of amino groups, carboxyl groups, and indole groups was carried out independently. In the absence of NAG3, the carboxyl group in Asp 101 was modified by glycinamidation, and the indole group in Trp 62 was modified by Koshland reagent. In the presence of NAG3, the degree of modification of Asp 101 and Trp 62 decreased. It is suggested that Asp 101 and Trp 62 are involved in the interaction with NAG3. The result is consistent with the one obtained by x-ray crystallography. It is indicated that the combination of chemical modification and mass spectrometry may be effective for the investigation of the binding reaction of enzyme to inhibitor and of protein-protein interaction.

Amines↗
PubMed1981

Postnatal modifications of the dendritic tree of cells in the inferior colliculus of the cat. A quantitative Golgi analysis.

Postnatal modifications of dendrites have been quantitatively studied by network analysis of the dendritic tree in the two central nuclei of the inferior colliculus in the cat. This analysis revealed cells with two types of branching patterns suggesting two different modes of growth. The predominant pattern is characterized by dichotomous branching on random segments (DR cells). However, a purely collateral branching pattern is particular to certain cells (CB cells). These two branching patterns were found in both nuclei of the IC in adult and young cats, but the exact significance of these two cell types remains unclear. The dendritic trees of cells in kittens differed from those of the adult cat. Also, the types of modification were different in the two functionally distinct nuclei of the inferior colliculus that we studied. The most dramatic modifications were observed in the dendritic tree of DR cells in the central nucleus, which receives fibers from the auditory nuclei in the brainstem. Two parameters were modified: the mean number of terminal segments and the mean total length of segments. Both parameters increased in the sagittal plane and decreased in the frontal plane. These modifications indicate a reorientation of the dendritic tree in the sagittal plane, along the incoming axons from the auditory nuclei. As these afferents become functionally mature only after birth, this spatial remodeling of the dendrites seems closely related to functional maturation of secondary auditory axons. In the dorsomedial nucleus that receives fibers from the auditory cortices, the dendritic tree of DR cells also undergoes spatial reorientation. This is more evident in the horizontal plane and with respect to the incoming axons. Our results suggest that the characteristic orientation of the dendritic tree of cells observed in the inferior colliculus of the adult cat is established only after the first postnatal weeks. This orientation seems to result from an active process of remodeling concomitant with the functional maturation of afferents, a fact already established for various cell types in the nervous system.

Afferent Pathways↗
PubMed1976

Modifications of the thymocyte membrane during redistribution of concanavalin A receptors.

The modifications of the thymocyte membrane which are induced by concanavalin A (Con A) were studied by means of the variation of electrophoretic mobility which the lectin induces on the cell. The electrophoretic mobility of thymocytes is increased when Con A is used under conditions expected to induce a cap formation and the subsequent endocytosis. This increase persists as long as the lectin is present in the medium and disappears as soon as the lectin is eliminated. The redistribution of the Con A receptors into spots and caps may partially explain this electrical modification. The ionized groups of the thymocyte membrane are drastically modified during these phenomena with: 1) a diminution by 61% in the density of the sialic carboxyl groups, 2) a decrease of 40% in the density of phosphate groups and of 60% in the density of amino groups, 3) a 20 times higher density of unidentified negatively charged groups. The electrophoretic mobility of normal human blood lymphocytes is similarly increased by Con A. A marginal difference exists in the shape of the dose response curves obtained when normal and cancerous thymocytes react with increasing doses of Con A. No measurable electrical modification was observed during redistribution of H-2 and Thy-1.2 antigens on EL4 cells. Some experiments suggest a possible correlation between the mitogenicity of the lectin and a measurable modification of the cell electrophoretic mobility.

Animals↗
PubMed1998

Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry.

In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.

Alkylation↗
PubMed2003

Surface modification of poly(dimethylsiloxane) microchannels.

This review looks at the efforts that are being made to modify the surface of poly(dimethylsiloxane) (PDMS) microchannels, in order to enhance applicability in the field of microfluidics. Many surface modifications of PDMS have been performed for electrophoretic separations, but new modifications are being done for emerging applications such as heterogeneous immunoassays and cell-based bioassays. These new modification techniques are powerful because they impart biospecificity to the microchannel surfaces and reduce protein adsorption. Most of these applications require the use of aqueous or polar solvents, which makes surface modification a very important topic.

Adsorption↗
PubMed2006

MUC2 expression is regulated by histone H3 modification and DNA methylation in pancreatic cancer.

Mucins are highly glycosylated proteins that play important roles in carcinogenesis. In pancreatic neoplasia, MUC2 mucin has been demonstrated as a tumor suppressor and we have reported that MUC2 is a favorable prognostic factor. Regulation of MUC2 gene expression is known to be controlled by DNA methylation, but the role of histone modification for MUC2 gene expression has yet to be clarified. Herein, we provide the first report that the histone H3 modification of the MUC2 promoter region regulates MUC2 gene expression. To investigate the histone modification and DNA methylation of the promoter region of the MUC2 gene, we treated 2 human pancreatic cancer cell lines, PANC1 (MUC2-negative) and BxPC3 (MUC2-positive) with the DNA methyltransferase inhibitor 5-azacytidine (5-aza), the histone deacetylase inhibitor trichostatin A (TSA), and a combination of these agents. The DNA methylation level of PANC1 cells was decreased by all 3 treatments, whereas histone H3-K4/K9 methylation and H3-K9/K27 acetylation in PANC1 cells was changed to the level in BxPC3 cells by treatment with TSA alone and with the 5-aza/TSA combination. The expression level of MUC2 mRNA in PANC1 cells exhibited a definite increase when treated with TSA and 5-aza/TSA, whereas 5-aza alone induced only a slight increase. Our results suggest that histone H3 modification in the 5' flanking region play an important role in MUC2 gene expression, possibly affecting DNA methylation. An understanding of these intimately correlated epigenetic changes may be of importance for predicting the outcome of patients with pancreatic neoplasms.

Azacitidine↗